Replication of undifferentiated cells in a weightless environment, uses thereof and facility for such replication and the acceleration of the evolution of plants and animals

ABSTRACT

Manufacturing processes are described for biological replication of undifferentiated plant and animal cells and tissue in a weightless condition, including those systems used in current stem cell research and development and use of undifferentiated parenchyma in plants. Additionally, methods for adapting plants and animals to survive outside their native environments are described. In particular, undifferentiated cells from plants or animals are replicated under weightless conditions in which cell replication or proliferation is accelerated and sustained. Under such conditions, the undifferentiated cells can be “forced” to express sets of genes useful for survival in particular environmental conditions. In this manner, cells surviving prolonged exposure to specific environmental conditions can be selected for and cultivated to produce an organism adapted to that particular environment in an accelerated manner. Methods of identifying specific genes associated with adaptation of a plant or animal to a specific environment are also described.

BACKGROUND OF THE INVENTION

A weightless condition on space orbit has produced many effects onvisitors to that region. Gravity still is present on orbit and throughthe course of interplanetary travel, but normal plant and animalfunctions as is known on Earth do not function in the same manner. Ifgravity acts as a dominant force in a weightless condition, then thereproduction of cells would follow the normal or close to normal patternexperienced on Earth. Evidence indicates that it is weightlessness andnot gravity that drives the biology of both plants and animals on spaceorbit and during interplanetary transits and human travel.

Weight is a physical attribute and force parameter. Weight is acondition wherein the “escape force” [as a function of the angularvelocity of a mass (angular momentum)] around a gravitational mass isless than the specific value of the gravitational force. That is, when abody is at rest on the Earth (stationary), it has an angular velocitydue to the Earth's rotation. This angular velocity opposes, to someextent, the gravitational force that draws the mass to thecenter-of-gravity point of Earth. As the angular velocity of the massincreases, the inertial force that opposes the gravitational forceincreases, to the extent that a sufficient increase will result in thegravitational force equaling the inertial force.

This is the case for masses that have been launched from Earth and arein an orbit of equilibrium around the Earth, i.e. “on orbit”. If theEarth had no angular velocity (i.e.—it did not rotate), the weight of amass would be greater than for a mass on a rotating Earth. It should benoted that the Earth's rotation has only a small effect on “gravity”(actually on “weight”), about 0.5%.

Weight is a condition that results from either (a) the presence of amass within a gravitational field and/or (b) a mass that is subjected toan external force that accelerates that mass (inertial gravity). Weight,as described in (a) or (b), results from a “mass-acceleration” force souniversal and common on Earth that it is normally perceived as anever-present attribute, state, or condition and is not included in manypatent process and protocol parameter descriptions. “Weight” is a normalcondition and physical parameter that affects “Living Systems” on Earth.Perceived weightlessness is experienced on orbit or in orbitingspacecraft that have reached a constant velocity on orbit or en-route tointerplanetary destinations.

Biological organisms, and specifically most higher-ordered biologicalorganisms, including plants, animals, including humans, hereafterreferred to as “High Order Living Biological Systems” (HOLBS) areadapted to Earth's gravity. The effects of weightlessness on plants andanimals are expressed by physiological effects that alter the physiologyand the morphology of the HOLBS, causing deleterious, irreversible,compromising, and transmuting effects from exposure to such conditions.For example, microgravity has been shown to have an impact on anastronaut's body in space. The effects of gravity in plants and animalsand the biological mechanisms involved in adapting to weightlessness maybe studied under real microgravity conditions. For example, research onastronauts has shown that body function is disturbed in microgravity.Space agencies are therefore continuing their research in hope ofeventually reducing or eliminating some of these undesirable physicaleffects that appear during a stay in space.

Generally, exposure of living systems to weightlessness results inbiological degradation. This degradation is a result of biologicalprocesses that have been fundamentally altered due to the absence of anessential force, gravity that is an essential component of thosebiological processes. Prolonged exposures to a weightless environmentcorrelate to increased biological alterations and degradations. It isthe attribute of “weightlessness” or “an apparent absence of gravityinduced-force” and/or the “absence of inertially-produced force” that isthe critical physical parameter which affects physiological process ofliving systems in a weightless environment.

On-orbit environments, e.g., as would be found on the InternationalSpace Station (ISS), are referred to as zero-g, zero-gravity, andgravity-free environments. These terms are misleading and incorrect. Theterm “weightless” is more correct and can be equated to “micro-gravity”for our purpose.

The Earth's “gravitational field,” whether in LEO, GEO or other orbits(600 km altitudes, etc.), is still present, i.e.—90+% of thegravitational field and gravitational force amplitude remains. Moreprecisely—static, on-orbit environments exhibit “weight-free”conditions, wherein gravity forces remain substantial as a consequenceof a continuing presence of Earth's gravitation field. For example:

-   -   On Earth at Sea Level: Assumption: Gravity=1.00, Weight=1.0.    -   On Orbit* at 600 km: Then Gravity=0.98, Weight=0.0. *Static        environment: zero local acceleration.

All mass, including the space vehicle and pay load will be at zeroweight on a earth bound scale that measures “weight”. However, it shouldbe noted that gravity is still present. The force of gravity between aspacecraft and Earth is directly proportional to the product of theirmasses and indirectly proportional to the square of the distance betweenthem. Acceleration of a mass into orbit overcomes the force of gravityand the mass will enter what is considered to be a “free-fall” effect.The mass in orbit may be a combination of many objects (masses) thatappear to be weightless in relation to the other masses in an apparentweightless “free-fall” environment. Gravity forces are still present,but the HOLBS are not able to function properly without a force thatmimics gravity.

As noted above, HOLBS exhibit marked physiological and biologicalchanges when resident on-orbit, e.g., given that this environment iswhere Gravity=0.95-1.00 and Weight=0.00-0.10, it is concluded that it isthe attribute of “weightlessness” that links these activities andprocesses, and that it may appear to be constant without regard togravity.

More specifically and to further clarify, it may be stated that onEarth, all higher-order living systems, plants, animals, includinghumans, proceed with biological processes under the influence of a“constant acceleration of their mass.” On Earth, this constantacceleration is a result of Earth's gravity, and the endless, largelyconstant, angular acceleration associated with Earth's gravitationalforce. It is recognized that there are slight (=<0.5%) variations ingravitational forces and Earth rotation angular velocities that occurdepending upon the location and region the mass is on the Earth.

On orbit, in a static environment (no apparent inertial acceleration ispresent and angular velocity is relatively constant),near-weightlessness (commonly referred to as zero-gravity ormicro-gravity) conditions are achieved. These terms are misleading andthe terms should be: zero-weight or micro-weight or some equivalents.The term “weightlessness” is being used herein to collectively refer tothese conditions. It is therefore evident that it is the attribute of“weightlessness”, not gravity, that is critical to active biologicalprocesses and components of plants, animals, humans, and higher-orderliving systems.

Furthermore, it is evident that commonly referred to conditions such as“hyper-gravity,” are misnomers, as the term has been associated with theforces resulting from the use of centrifuges that create “artificialgravity”, when in actuality, they produce centripetal, angularacceleration forces that are more accurately “inertia produced angularaccelerations,” and may be viewed as or termed “inertial gravity” ormore accurately “hyper-weight”. Linear accelerations also apply here: inthe form of artificial gravity, especially on long space flights in theacceleration and de-acceleration phases. In summary, an angular (orlinearly) induced acceleration of a mass will result in a force uponthat mass that causes that mass to possess “weight”. Weight is anattribute of acceleration of mass (evidenced elementarily by its unit ofmeasure being in—meters/per second/per second).

Thus, it is submitted that a linkage exists between mass-acceleration(as a mass-energy function) and mass-density (as a mass function), andthat the linkage of these two attributes has specific direct andindirect effects upon certain biological processes that occur in plantsand animals, including humans and higher-order living systems (HOLBS),where these effects impact functions of cellular replication,reproduction, regeneration, creation, differentiation, specialization,function, cell life-span, suspended animation, and cell death.

It is evident that specific, fundamental biological processes possessedby plants, animals, humans and higher-order organisms and living systemsand their growth, development, and life-cycles are affected byweightlessness, and, that these living processes differ critically andprofoundly when those processes occur in a “weight” (mass-accelerating)environment versus a “weightless” (mass-zero-accelerating) environment.Therefore it is submitted that it is the attribute or characteristic of“weightlessness” that determines and assures and drives certainessential plant, animal (including humans) and higher-living systemprocesses and not the presence of gravity, nor the presence of agravitational field or its effects.

Single celled organisms such as bacteria reproduce with littledifferentiation unless there is a mistake in the normal cellsdevelopment that may produce a new strain. Bacteria can reproduce atamazing rates. Bacteria are protected by a cell wall that surround theprotoplasm. The asexual reproduction of the bacteria continues untilregulated by outside forces. Weightless conditions do not appear to be afactor in the reproduction of single celled bacteria over at least onegeneration.

The replication of undifferentiated cells from both plants and animalsmay also follow the same model as the yeast if proper preparation,transport and retrieval process are followed for the production ofundifferentiated cells on orbit.

Experiments and the data derived from visitors to weightlessnessindicate that weightlessness plays a role in the development of humancells. See e.g., Longnecker et al., ed., “Review of NASA's LongitudinalStudy of Astronaut Health,” Jan. 20, 2004, The Institute of Medicine,which is herein incorporated by reference in its entirety. The Earth'sgravitational force influences the developing cells to differentiateinto specialized cells, which in plants may be branches or roots and inanimals the brain or the legs. In weightlessness, the differentiation ofthe primordial cells in both plants and animals cannot occur. Thecontinuous reproduction of the primordial tissues will continue with theright nutrient system producing undifferentiated cells in both plantsand animals until terminated.

There are techniques that work on a limited basis for both plants andanimals in a gravity environmental condition. The constraints areenormous and only a limited amount of primordial cells are available.The use of a manufacturing process that can produce an unlimited supply(based on logistics) of undifferentiated plant and animal cells can beachieved using zero gravity during the replication of undifferentiatedtissues. The present invention recognizes the effect of weightlessnesson animal and plant cells and utilizes this recognition to provide amethod for replicating large quantities of undifferentiated cells forboth animals and plants. Additionally, the present invention comprises aspace-based on-orbit manufacturing facility for creation, replication,production, storage and ultimate transport of undifferentiated stemcells from a weightlessness environment to a gravity environment onEarth or at another facility. Currently used space vehicles can be usedfor the replication of stem cells and undifferentiated plant cells.

The plant studies described herein and conducted on the spacetransportation system (STS)-118 mission demonstrate cell replicationrather than cell differentiation and that the cells replicating in spacedemonstrate a greater mass than the control cells on Earth, therebydemonstrating replication of undifferentiated cell in weightlessness.These results allow a further expansion of the basic logic to includethe acceleration of gene expression in undifferentiated cells on orbitand using the protocol discussed herein to provide for a acceleration ofthe natural selection process for plants and animals.

The accelerated and sustained proliferation of undifferentiated cells onorbit provides an opportunity to “force” the cells to express genes thatwill enable them to adapt to specific environmental conditions. In otherwords, imposing environmental constraints on the undifferentiated cellswhile they are proliferating will result in a subset of cells expressingthe necessary genes which enable the cells to survive in that specificenvironmental condition. One can then cultivate an organism from theseselected cells that will survive in that particular environmentalcondition. For example, undifferentiated cells from a species of citrusplant can be propagated at cooler temperatures on orbit. Cells survivingthe cool temperature conditions can be returned to earth and cultivatedto produce a citrus plant than can thrive in cold temperatureenvironments.

These techniques are applicable to both plant and animal cells. “Hardy”organisms can be produced by selecting undifferentiated cells expressinggenes for survival in particular environments, including extremeenvironments, such as the surface of Mars. The present inventionrecognizes the advantages of replicating primordial cells on orbit, andutilizes these advantages to expand the range of cell function thusaccelerating the evolution of organisms. Therefore, the presentinvention provides methods for adapting plants and animals to surviveoutside their native environments.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides a method of using at leastone undifferentiated cell as a result of uniting two haploid cells toform a diploid single cell under conditions to support fertilization ina weightless condition or environment, wherein said the diploid cell canreplicate itself continuously to provide a mass of undifferentiatedcells.

In one embodiment, the present invention comprises uniting a sperm andegg from an animal or pollen and ovary from a plant to form a singlecell in a weightless condition.

In another embodiment, the present invention further comprises unitingtwo haploid cells, such as a sperm and egg from an animal or pollen andovary from a plant to form a single cell in a weightless condition orenvironment, and culturing the single cell to replicate in a weightlesscondition or environment.

In yet another embodiment, the present invention additionally comprisesuniting two haploid cells, such as a sperm and egg from an animal orpollen and ovary from a plant to form a diploid single cell in a gravitycondition or environment, such as under normal Earth gravity, afterunification or fertilization is complete, immediately preserving coolingto below metabolic activity) the resulting single cell so that noreplication of the single cell occurs, then under weightless conditions,reviving the single cell, and culturing the single cell in a weightlesscondition or environment to obtain replication of the single cell.

In yet another embodiment, the present invention also comprises adiploid single cell, such as a fertilized egg or single cell produced byuniting a sperm and an egg in an animal or pollen and ovary from a plantunder conditions to support fertilization in a weightless environment orcondition.

In one embodiment, the present invention further comprises one or moreundifferentiated diploid cells produced by uniting two haploid cells,such as a sperm and an egg from an animal or pollen and ovary from aplant under conditions to support fertilization resulting in afertilized egg or diploid single cell in a weightless environment orcondition and by culturing the fertilized egg in a weightlessenvironment or condition resulting in the replication of the fertilizedegg so that one or more undifferentiated cells are replicated.

In another embodiment, the present invention further comprises the useof already undifferentiated tissues from liver, kidney, etc. of animals(including humans) properly preserved (cooling, etc.) for transport toon orbit for replication.

In yet another embodiment, the present invention encompasses the use ofundifferentiated plant tissues for the purpose of replication of theundifferentiated cells on orbit en masse.

In another aspect, the present invention additionally comprises a methodof administration of the undifferentiated cells to a subject or patientin need of treatment by the undifferentiated stem cells, wherein thecells are produced by uniting two haploid cells, such as a sperm and anegg from an animal of the same blood type to support replication of thesame tissues or from replication of the undifferentiated tissuesproduced on orbit.

In some embodiments, the present invention additionally comprises knownmanufacturing, processes, biological processes, cellular, organism,organ, and living-systems development, creation, fabrication,harvesting, growth, replication, reproduction, in-vivo, in-vitro, orequivalent activities, suspended animation processes, developmentactions, or processes that are undertaken in a weightless environment,or more specifically in a mass-acceleration environment.

In another aspect, the present invention provides structures andprocesses for on-orbit vehicles, systems, spacecraft, space ships, andrelated habitats and containments used to transit to and/or travel todistant destinations, wherein those structures and processes resident orpart of those structures are configured in a fashion, such that theywill lower, reduce, mitigate, or eliminate the biological degradation ofplants and animals (including humans) in space, in weightlessenvironments, on-orbit, during inter-planetary transits or excursions,and when resident on sub-G environments (Moon, Mars, etc.).

In yet another aspect, the present invention provides methods ofadapting a plant or animal to grow in a hostile environment or anenvironment outside of the plant's or animal's native environment. In apreferred embodiment, the method comprises culturing undifferentiatedcells from a plant or animal in a weightless condition that mimics atleast one element of the hostile or non-native environment to which theplant or animal is to be adapted; selecting the cells that replicate insaid condition; and cultivating said selected cells to produce plants oranimals, wherein the plants or animals are adapted to grow in saidhostile environment.

In one embodiment, the method further comprises evaluating the resultantplants or animals in the hostile environment. The organisms may beevaluated on various criteria including length of survival, growth rate,reproductive capability, cell structure, hardiness in hostile ornon-native environments and other gene expressions including but notlimited to those enumerated above.

Various environmental stimuli can be used in the methods of theinvention to induce the adaptation of the plant or animal to the hostileenvironment. These stimuli may include excessive heat, excessive cold,low barometric pressure, excessive radiation, high carbon dioxidelevels, low humidity, high humidity, drought conditions and duration ofsunlight exposure and other environmental factors to mimic conditions ina climate other than the present native climate of the plant or animal.

The present invention also provides methods of identifying genesassociated with adaptation of a plant or animal to a hostileenvironment. In one embodiment, the method comprises culturingundifferentiated cells from the plant or animal in a weightlesscondition that mimics at least one element of the hostile environment towhich the plant or animal is to be adapted; selecting the cells thatreplicate or proliferate in said condition; examining the geneexpression profile of the selected cells in comparison to the geneexpression profile of control cells; and identifying genes that have achange in expression level, wherein the identified genes are associatedwith adaptation to the hostile environment. The selected genes that aredifferentially expressed in the various environmental conditions can befurther used to produce transgenic plants and animals with the desiredadaptive characteristics by introducing these genes into cells thatmature into plants or animals. Through this process, the adaptationand/or evolution of plants and animals can be accelerated.

In another embodiment, the method can be used for the production ofvaccines to be used in animals and humans. By stressing pathogenicmicroorganisms on orbit, modified strains of microorganisms can beproduced. These can include less virulent and/or more virulent strainsof bacteria and viruses, which can then be utilized for the productionof improved vaccines.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. A 3-dimensional on-orbit manufacturing facility useful formanufacturing stem cells and stem cell tissues in a weightlessenvironment.

FIG. 2. A robotic processing system for on-orbit manufacturing of plantand animal undifferentiated cells useful in the present invention.

FIG. 3. Gene expression profiles of Arabidopsis thaliana seedlings onorbit and on Earth. The gene expression pattern from orbit is plottedversus the gene expression pattern on Earth (panel A). Panel B depicts asubset of the gene expression profile to illustrate heat-shock and CAB(light) genes. The lines labeled with 4× and 10× illustrate four-foldand ten-fold threshold levels of expression, respectively.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides manufacturing processes for biologicalreproduction of plants and animals in a weightless condition, includingthose systems used in current stem cell research and development and useof undifferentiated parenchyma in plants.

In one embodiment, the present invention provides a method ofreplicating at least one undifferentiated cell comprising uniting twohaploid cells to form a diploid single cell under conditions to supportfertilization in a weightless condition or environment, wherein saiddiploid single cell can replicate itself forming another diploid celland wherein said diploid single cells will not develop into an embryo ofdifferentiated cells and tissue. The two haploid cells may be gametes,and more specifically the gametes are of the opposite sex or matingtypes.

The present invention includes the use of the purest form ofundifferentiated tissues of organs such as, but not limited to liver andkidneys that are replicated on orbit. The present invention comprisesuniting a sperm and egg from an animal or pollen and ovule from theovary from a plant to form a single diploid cell, such as zygote, in aweightless condition or environment as defined herein. The single cellwill not divide to ultimately produce differentiated cells with thecapability of developing into an embryo when created or produced orcultured under a weightless environment. Then the replication of cellsfrom newly united sperm and egg or pollen and ovary in a weightlesscondition provides an endless supply of undifferentiated cells andtissues that can be used to reproduce cells that are identical to theparent cells without transformation into multi-celled, differentiatedtissues. One or both of the haploid cells utilized in the method maycontain an incomplete haploid genome of the cell, and could be missinggenetic material or genes or fragments thereof.

The present invention further comprises uniting two haploid cells, suchas a sperm and egg from an animal or pollen and ovary from a plant toform a single diploid cell in a weightless condition or environment, andculturing the single cell to replicate into undifferentiated orprimordial cell and tissue in a weightless condition or environment. Themethod further comprises culturing the diploid single cell to obtainmultiple replications of the replicated single cells to a suspension ofundifferentiated cells. The method further comprises harvesting theundifferentiated cells from the suspension. Additionally, the methodfurther comprises changing the culture conditions of the suspension orharvested undifferentiated cells to a state of suspended animation orquiescence.

The present invention further intends to encompass a method of producingor replicating at least one undifferentiated cell comprising uniting twohaploid cells to form another diploid single cell under conditions tosupport fertilization in a gravity condition or environment under normalearth gravity, after unification is complete, preserving the resultingdiploid single cell so that no replication of the diploid single celloccurs, reviving the diploid single cell under a weightless condition orenvironment, and culturing the diploid single cell in culture medium ina weightless condition or environment to obtain replication of thediploid single cell that will not develop into an embryo ofdifferentiated or primordial cells and tissue. The two haploid cells maybe gametes, and more specifically the gametes are of the opposite sex ormating types. Further, the gametes are a sperm and an egg from an animalor a pollen and an ovule from an plant. The diploid single cell can be azygote. Additionally, at least one of the haploid cells comprises anincomplete haploid genome of said cell.

The method further comprises culturing the diploid single cell toreplicate in a weightless condition or environment. Further, the methodfurther comprises culturing the diploid single cell to obtain multiplereplications of the replicated singles cells to a suspension ofundifferentiated cells. The method further comprises harvesting theundifferentiated cells from the suspension, and optionally, changing theculture conditions of the harvested undifferentiated cells to a state ofsuspended animation or quiescence. Or alternatively, the method furthercomprises changing the culture conditions of the suspension ofundifferentiated cells to a state of suspended animation or quiescence.Also encompassed by this method, is an isolated diploid single cellproduced by the methods described herein. This single cell can be azygote. Additionally, a composition comprising more than one single cellproduced by the methods described herein is disclosed that is optionallymixed with a pharmaceutically acceptable carrier.

The present invention further comprises uniting two haploid cells, suchas a sperm and egg from an animal or pollen and ovule from the ovary ofa plant to form a single diploid cell in a weightless condition orenvironment, and culturing the single cell to replicate intoundifferentiated or primordial cell and tissue in a weightless conditionor environment.

The method further comprises culturing the diploid single cell to obtainmultiple replications of the replicated single cells to a suspension ofundifferentiated cells. The method further comprises harvesting theundifferentiated cells from the suspension. Additionally, the methodfurther comprises changing the culture conditions of the suspension orharvested undifferentiated cells to a state of suspended animation orquiescence.

The present invention also comprises a fertilized egg or single cellproduced by uniting a sperm and an egg in an animal or pollen and ovaryfrom a plant under conditions to support fertilization in a weightlessenvironment or condition. An isolated diploid single cell produced bythe method described herein, and wherein the diploid single cellcomprises a zygote which can be replicated without development intofurther stages.

The present invention further comprises a method producing at least oneundifferentiated cell comprising uniting a sperm and egg of an animalprior to culturing in a weightless condition or environment to produceundifferentiated cells that replicate at a higher rate than in a weightor gravity condition or environment.

Also encompassed by the present invention is a composition comprisingmore than one single cells (diploid) of produced by the method describedherein, and optionally a pharmaceutically acceptable carrier. Such adiploid single cell comprises a zygote that will not progress into afurther stage of development unless prompted to do so.

The present invention further comprises one or more undifferentiatedcells produced by uniting a sperm and an egg from an animal or pollenand ovary from a plant under conditions to support fertilizationresulting in a fertilized egg or single cell in a weightless environmentor condition and by culturing the fertilized egg in a weightlessenvironment or condition resulting in the replication of the fertilizedegg so that one or more undifferentiated cells are produced orreplicated.

The present invention additionally comprises a method of administrationof an effective amount of the undifferentiated cells produced by themethods of the present invention to a subject or patient in need oftreatment by the undifferentiated cells, wherein the cells are producedby uniting two haploid cells, such a sperm and an egg from an animal orpollen and ovary from a plant under conditions to support fertilizationto for a fertilized egg or single cell in a weightless environment orcondition and by culturing the fertilized egg in a weightlessenvironment or condition resulting in the replication of the fertilizedegg so that one or more undifferentiated cells are produced orreplicated. The subjects treated may be in need of undifferentiatedcells to treat diseases and conditions, such a type I diabetes,Parkinson's disease, Alzheimer's disease, blood diseases, such asleukemia, different types of anemia, systemic lupus, autoimmune diseasesand deficiencies, heart tissue repair, bone and cartilage repair, eyeand skin repair to name a few of the conditions and diseases that canbenefit from the undifferentiated stem cells produced by the presentinvention. The stem cells will be administered utilizing known methodspresently used for the administration of adult stem cells to patients.Known pharmaceutically acceptable carriers can be mixed with thecomposition comprising the undifferentiated stem cells produced by themethods of the present invention under weightlessness conditions orenvironments to form a pharmaceutical compositions comprising theundifferentiated stem cells and optionally a pharmaceutically acceptablecarrier.

The present invention additionally comprises manufacturing processes,biological processes, cellular, organism, organ, and living-systemsdevelopment, creation, fabrication, harvesting, growth, replication,reproduction, in vivo, in vitro, or equivalent activities, suspendedanimation processes, development actions, or processes that areundertaken in a weightless environment, or more specifically in amass-acceleration environment wherein the mass-velocity is near-constantand the mass-acceleration is near-constant and is of a value which isequal and opposite to the force-value of gravity present.

The present invention further comprises processes, manufacturing,development, prototyping, activities and actions that are directed tothe development, replication, reproduction, growth, maturation, orcreation of new cellular materials, tissues, cellular components,organs, living systems, and suspended animationtechnologies—specifically when those activities are in a near weightlessor fully weightless condition.

The present invention also comprises a culture device for replicating adiploid cell produced by the methods disclosed herein and a method formanufacturing, processing, storing (including suspended animation) andshipping cells created by the unification of animal (sperm and egg) andplant (pollen and ovary) for the purpose of replication of primordialalso known as undifferentiated cells and tissues, including, but notlimited to animal, such as human, stem cells and plants(undifferentiated tissues such as apical meristems), and other tissues,specifically being methodologies that utilize processing environments,wherein, the forces of weigh are absent, specifically near-weightlessand fully-weightless environments and processing conditions.

The present invention additionally comprises those structures andprocesses for on-orbit vehicles, systems, spacecraft, space ships, andrelated habitats and containments used to transit to and/or travel todistant destinations, wherein those structures and processes resident orpart of those structures are configured in a fashion, such that theywill lower, reduce, mitigate, or eliminate the biological degradation ofplants and animals (including humans) in space, in weightlessenvironments, on-orbit, during inter-planetary transits or excursions,and when resident on sub-G environments (Moon, Mars, etc.).

All of the manufacturing processes, biological process, and relatedactions and activities conducted on Earth, are subject to and include a“mass-acceleration” component. A forcing component that results in acondition or characteristic known as “weightlessness” is encompassed bythe present invention.

Gravity is not the direct force or condition that drives biologicalprocesses in a weightless environment. Gravity on Earth, for example, isthe condition that results in forces that drive the biological universeon Earth. Gravity is not absent on-orbit. Substantial gravitationalfields exist on-orbit and in space. The gravity on orbit isapproximately 95% the gravity on Earth and approximately 0% weight. Therules regarding reproduction of cells change in a weightless condition.Both plant and animal cells do not replicate in a weightless conditionaccording to the rules on Earth. On Earth, gravity is a condition thatties to specific forces that govern the reproduction of cells.

Gravity is a condition which may contribute to “weightlessness” or“weight” depending upon the presence of other forces, i.e.—otherconditions, energies, or forces which result in the acceleration of masspresent, and which may add to or oppose the effects of mass-accelerationthat result from the presence of mass within a gravitational field. In aweightless condition (where the acceleration of mass is generally equalto and opposite the force that exists from gravitational conditions),gravity is a null-component of the system and does not have a unique,essential, critical, or driving effect upon the biological processesdescribed and claimed herein.

To clarify the present invention with respect to gravity, gravity forcesand gravitational fields, it is recognized that gravitational fields andforces exist to varying degrees and at varying amplitudes on Earth, inthe Earth, at altitudes above the Earth, on-orbit around the Earth,between Earth and Moon, and inter-planetary in the Solar System andbeyond. The gravitational field forces in these regions vary fromapproximately 1 G on Earth, to 0.4 G, to 0.6 G on Mars, etc., and atother varying amplitudes throughout the Solar System. It is recognizedthat these gravitational force scalars (values) remain, whether or notother inertial forces are present. It is recognized that gravitationalforces at any particular point within the Solar System, vary temporallygiven the motions of the planets and heavenly bodies present within theSystem. It is recognized that gravitational forces present on localsystems, e.g.—Earth, Moon, Mars, are relatively constant and that localgravitational field amplitudes in these point regions are relativelyconstant in amplitude over time.

The present invention recognizes that an essential and criticalconstellation of processes associated with the reproduction of cells isuniquely and profoundly different in a weightless condition, than wherethose processes are conducted or performed or executed in an environmentwhere (a) gravity that results in mass-acceleration exists, (b) onEarth, (c) in artificial gravity environments [i.e.—inertial gravityenvironments e.g.—centrifuges, so-called hyper-gravity, more correctlytermed hyper-weight) systems, units, devices, or facilities.] The natureof all reproduction processes are influenced by gravity and cellscontinue to replicate under the influence of the gravitational force,following a genetic code prescribed in the genes that respond to theforce of gravity. A fertilized egg replicates, and begins forming unionsand congregate into a mass that expands exponential fashion (1, 2, 4, 8,etc.) to eventually form an embryo, then differentiated cells andtissue. The present invention is different from the development of thecells in the gravity environment, in that the weightless conditionsresult in the replication of cells to form cultures of undifferentiatedcells that do not develop into an embryo. In weightless conditions, afertilized egg can be replicated indefinitely to form cultures ofundifferentiated cells that do not develop into an embryo.

Methods of preparing such undifferentiated cell cultures in weightlessenvironments and their use are described herein. Plant experiments onthe STS-118 mission demonstrated that plants replicate in weightlessnessand since both plants and animals follow the Kreb's Cycle, animal cellssimilarly replicate in weightlessness.

The present invention further comprises the biological and living-systemprocesses, manufacturing, cellular, replication, reproduction, creation,duplication, harvesting, development, maturation, and growth, processingstorage (including suspended animation and shipping) that are conductedor performed, whether through human or autonomous-control, in (process)environments that have zero-acceleration of mass in orbit or ininterplanetary travel and/or where the acceleration of mass is equal andopposite the gravity induced force present.

To further clarify those process environments where there is nomass-acceleration force present and/or where the acceleration of mass isequal and opposite the gravity induced force present, nomass-acceleration force is included as part of the biological,physiological, replication, reproduction, growth, harvesting,manufacturing, or production operation on orbit or in interplanetarytravel unless artificially produced.

The present invention further comprises all “replication” processeswhere plants and/or animals including humans, and higher-order organismsand living systems “HOLBS” and the cellular processes of which they arebased-upon duplicate their cellular constituents, most specifically andcritically, those cellular aspects and types which are primordial instructure and biological configuration.

The present invention comprises the “replication processes” ascontrasted to normally occurring “reproductive processes” in orbit orduring interplanetary travel. Wherein those cellular activities thatprogress through normal maturation stages and cycles are known as“reproductive” on Earth, however, on orbit, cellular activities that“duplicate” and “replicate” copies of cells that are identical instructure and function to their originating predecessors is“replication” and the process, storage (including suspended animation)and shipping is encompassed by the present invention.

Wherein, specifically, the creation of, processing, and manufacturing of“primordial stem cells” from animals, undifferentiated parenchyma fromplants and from other primordial tissues are central to the presentinvention, and where these processes are defined and termed as being“replication”, not reproductive.

The present invention further comprises processes that occur or areperformed in zero-acceleration of mass environments and/or where theacceleration of mass is equal and opposite the gravity induced forcepresent. In one embodiment, the present invention comprises processeswhere the production of “undifferentiated tissues, stem cells—of anystage or type or maturation, of primordial cells, of zygotes, sperm,egg, pollen, are part of said processes, and particularly wherein theobjective is the manufacture, creation, growth, development, harvestingor production of these cellular masses, tissues, organisms, and/orsimilar living systems.

The present invention comprises processes that enable the “replication”of a single cell from newly united sperm and egg or pollen and ovary ina weightless condition that under continued weightless conditions willprovide an endless supply of undifferentiated cells or tissues that canbe used to reproduce cells that are identical to the parent cellswithout transformation into multi-celled tissues or differentiatedtissues.

In animals and plants, the sexual stage of the organism is one celledfor the male (sperm or pollen) or female (egg or ovary). Both the sexualstage of plants and animals are also protected by cell walls. Such cellscan be transported into orbit without interference for use in thereplication phase of undifferentiated cells. The selection of the cellsbeing used for replication must be conducted with great care to ensurethe germ plasma will be a match with the use intended. For example,matching blood types from a donor cell with the blood types of aneventual recipient is a criteria. The plant and animal single-celledmale and female sexual stages are also single celled and are noteffected by gravity in limited experimentation. Formation of a zygoteoccurs after the union of the egg and sperm and exists up until thepoint that the cell replication begins. It is at this stage that thecells in either plants or animals can differentiate into any part of theplant or animal.

Furthermore, the present invention comprises all manufacturing processesfor biological replication and reproduction of undifferentiated cells ofboth plants and animals in a weightless condition.

The present invention also comprises all processes and systems used incurrent stem cell research, development, processing, and manufacture,but with the additional step or condition of performing or conductingthese processes in a “weightless” (zero-acceleration of mass)environment as defined in the present invention and as understood bypersons skilled in the art of gravitational theories and sciences,and/or where the acceleration of mass is equal and opposite the gravityinduced force present.

By way of further clarification, the above-described invention comprisesall stem cell, cellular, tissue, biological entity, and living organismprocesses and processing and all processes and process patents for same,applied for, pending, filed, and issued that predicate their disclosedinventions on processes that are conducted either (a) on Earth or (b) inan environment wherein there is an energy, force, or equivalent processthat includes a significant “mass-accelerating” component or free-falleffect as previously described. However, the present inventiondistinguishes its embodiments from these known processes by performingall of the steps and processes including cultivation of or culturing theresulting replicated cells in a weightless environment. In other words,the disclosed or known process elements, contents and activities areconducted in an environment where the constituents (cells, tissues,organs, organisms) have weight (and where the origins or causes of thatweight may be from either gravitational or inertial energy sources),such as performed on Earth in its gravitational environment.

In a further embodiment, the present invention comprises the use ofundifferentiated parenchyma in plants and undifferentiated stem cells inanimals as a unique process component for plant and animal replicationfor mitigation of diseases or arthropods.

Biological systems concerning the replication of cells adjacent toreproductive tissues in mature animals or plants in a weightlesscondition do not follow the rules for a gravity condition, but ratherfollow a non-descript process that provides for replication ofprimordial tissues without differentiating into a conglomeration ofcells to form a plant or animal. Space visitors have reproduction ofcells that mimic but do not follow the normal reproduction into afunctional cell. The primordial cells adjacent to bone, muscle, nerveand other types of differentiated cells and the mass of cells thatproduces blood and hormones, do not function as in a gravity or otherforce field (accelerated mass), and form non-functional cells. Spacevisitors experience this effect and indicate that cell reproduction isnot normal. It is believed that the cells being reproduced areundifferentiated with minimal influence from the adjoining tissues witha result being non-functional tissue.

The primary cell to be used for stem cell replication is formed by theunion of a sperm and egg in animals and can be conducted in a weightless(in orbit) environment. In plants, the apical meristem cells(undifferentiated parenchyma) resulting from the union of pollen and anovary in a weightless condition can provide an unlimited supply ofidentical cells that do not differentiate at the time of replication.The present invention is intended to encompass all processes currentlyused and known by persons skilled in the art for the reproduction ofplants and animals in a gravity driven environment for use to replicatecells at the telophase stage of mitosis in a weightless condition.

The present invention is based on the premise that the biologicalsystems including plants, animals, humans, and high-order living systemsfunction in accordance with a newly coined term “K-Law,” which statesthat primordial cells and living tissues will “replicate,” which isdefined as producing an exact copy in a “zero-acceleration of mass”environment. The “K-Law” states that primordial cells formed inweightlessness will self-replicate, but will not proceed to the nextstep of cellular and tissue replication resulting in cellulardifferentiation into recognizable plants or animals, i.e.—progressivematuration and differentiation along a recognizable life-cycle path.Stem cell staging, for example, will progress from primordial tissues toadult stem cells, but will not combine with other stem cells to form azygote.

With regard to the present invention, the “K-Law” is that which isdefined by the conditions wherein primordial cells, in azero-acceleration of mass environment are allowed to develop and thatsaid development is not reproductive, but rather these cells onlyreplicate. The “K-Law” applies and is effective only in“zero-acceleration of mass” environments, or where there is a“net-zero-acceleration of mass” and/or where the acceleration of mass isequal and opposite the gravity induced force present. The presentinvention further encompasses all processes wherein the “K-Law” applies.

Furthermore, the present invention comprises those processes wherein theprimary cell formed by the union of a sperm and egg (in animals,including man) that are processed, produced or created in a weightlessenvironment, including manufacturing, handling, storage (includingsuspended animation) and shipping of the final product. And wherein, theprocess for plants is that which employs the apical meristem cells(undifferentiated parenchyma) resulting from union of pollen and anovary in a weightless condition, such that the result is an unlimitedsupply of identical cells that do not differentiate at the time ofreplication into specialized cells and tissue.

The present invention also comprises all processes currently used andknown for the reproduction of plants and animals in a gravity drivenenvironment for use to replicate cells at the telophase stage of mitosisin a weightless condition, storage (including suspended animation),handling and shipping of the final product. Further, primordial cellscreated or produced as a result of the unification of an egg and spermthat have or have not been compromised by another force (example,Earth's gravity) will reproduce identical cells (given mistakes ingenetic code) in a weightless environment. Additionally, primordialcells resulting from apical meristem tissue or from the germination of aseed and/or new tissues from another source (new supplies) willreproduce the same undifferentiated cells during mitosis in a weightlessenvironment. The present invention encompasses all known processes thatare Earth bound to reproduce cells at the telophase stage of mitosis forthe purposes of stem cell replication and/or other purposes but for usein a weightless environment.

The present invention further comprises all Earth bound processes forthe replication and manufacturing of undifferentiated tissues fromplants and/or animals or both for use in a weightless and/or reduced“gravity” condition. Such conditions include the replication ofundifferentiated tissues on the Moon, Mars, etc., or other reducedgravity conditions.

Additionally, the present invention also comprises an orbiting craft,for example, Space Station, to provide a weightless condition.Gravitational forces are still present on such a craft. However, thebiological process is not chained to gravity; but the condition ofweightlessness. The extensive literature describes the maladies sufferedby space visitor, especially in prolonged stays. The present inventionprovides a unique method of transfer of plants and animals from aweightless condition to a device that mimics the force of gravity onEarth and back again to weightlessness. This method of producing acondition that mimics gravity in an orbiting vehicle will allow animalsand plants to survive long periods of space flight.

Long-term visitors to weightlessness are afflicted by many deficienciesduring the stay in this environment, especially “wasting” of tissues ona Bell Curve—the longer the stay, the more “wasting” of the body andfunctions. Exercise machines, etc. have been incorporated intoprocedures to “condition” visitors to the weightless environment. Allhave failed to a lesser or greater degree. A “Wheel” circulating arounda central unit was impractical because the whole system would rotate.However, the present invention provides a “Clutch” system in which thehub of the system will be at a weightless condition, a secondary“Clutch” that can be stopped at the hub (weightless condition), allowingpersonnel and/or equipment to be off-loaded and then gain speed to thecirculating “Wheel” at intervals to allow for proper balance. Thepresent invention comprises such a vehicle comprising the abovedescribed “Wheel” and “Clutch” that will allow for weightlessnessexperimentation and/or allow for long periods of orbital and/or spacetravel to distance objective (Mars, etc.). The “clutch” and wheel can becomposed of composite section that can be transported to orbit andassembled. The “clutch” and wheel can also be constructed ofpuncture-proof inflatable composites that can be transported to orbitand inflated in two sections for the “clutch” and the wheel.(attachment). The number of “spokes” in the wheel can vary, but weightdistribution and balance must be of concern.

The present invention is also intended to encompass a system to be usedto develop artificial gravity while still maintaining a weightlesscondition for the reproduction of undifferentiated tissues of bothplants and animals.

The present invention further comprises the system as described can be“mated” with the current space station that can act as a haven for thecrew of the Space Station. A universal “mating” system to allow transferof supplies, personnel and experiments will be incorporated. The spacetourism possibilities could also utilize the use of the “clutch” andwheel concept, especially if the space visitor will be in orbit for sometime.

By way of further explanation of the present invention, replication ofcells in a weightless environment will not reproduce in a normal fashionbecause a gravitational force must be present to align the components ofthe primordial cells. For example, germinating seeds on Earth align theroots to go down and the stem to go upward due to the force of gravity.Newly united sperm and egg form zygotes. The present invention ispremised upon the theory that cells that are not influenced by a forcethat is required to properly align the components of a reproductivesystem will not act according to known understanding of common cellreplication. Cells that replicate according to the laws that governreplication will be replaced by a new set of rules (K Law) based on a“weightless” condition that allows a primordial cell to reproduce itselfover and over again.

Space visitors have suffered numerous maladies in direct proportion tothe time spent in orbit. The visitors are experiencing the result ofprimordial cell replication adjacent to existing cells where the “new”cells are dysfunctional because there is no force, such as gravity or anartificial gravity-like force, to provide alignment according to the“gravity” instruction. Cells adjacent to muscles, bone, etc. willattempt to reproduce the same cells, but instead produce a cell thelooks like the original cell, but does not function properly because thegravity like force was not there to align the components of the cell inproper fashion (K Law). The cells that replicate in space visitors areprimordial in nature, and cannot provide tissues adjacent to the tissue(bone, muscle, nerve, blood cells etc.) which resemble the same tissuesin a Earth environment. Instead the cells produced in a weightlessenvironment will follow the instructions of the adjacent cell, but notreproduce totally viable cells that function as the cells would if onEarth. The references sited attest to the maladies that our spacevisitors have encountered and are consistent with the (K Law).

Primordial or Undifferentiated Replication in Weightlessness

However, the bad news can be the good news, and knowing the problem willallow a solution. As described herein, in a weightless condition, bothplants and animals, cells can replicate to the telophase stage ofmitosis and remain undifferentiated. Therefore, a weightless conditionallows the production of undifferentiated cells that can be harvested enmasse. Without the influence of a gravitational force, it is submittedthat cells do not replicate properly beyond the telophase stage ofmitosis to form a plant or animal. Cells at the telophase or final stageof mitosis in a weightless condition only replicate the original cell atthe telophase stage. Cells produced to the telophase stage of mitosiscan be harvested on a continuous basis and immediately stored in a stateof suspended animation for transport back to Earth or an out-post wherea suitable condition exists for reproduction.

Space Visitors

In a weightless environment, formation of cells adjacent to tissues in aspace visitor (red blood cells, bone, muscle, etc.) will form the samecell adjacent to the parent cell, but will be dysfunctional. A differentsystem, but an analogy, is a cell or cells in the body that become adifferent, but similar cell, because of lack of sufficient oxygen andbecause of not being oxygenated properly, the cells become anaerobic.Those cells follow a fermentation process and do not follow the “Krebs”cycle, and although the cells will form, they will not be functional.There is a high probability that those cells can become cancerous.

Cells adjacent to normally reproducing tissues (skin, bone, etc.) in aspace visitor in a weightless condition produce undifferentiated cellsthat would produce normal cells by instructions by the adjacent tissueand gravity. However, a weightless environment will produce cells thathave all the genetic make-up of the adjacent cells (muscle, skin, etc.),but will not be functional.

The basic problem with the reproduction of the primordial cells forcommercial purposes of both plant and animals has been a system thatcompensates for replication of the multi-celled higher plants andanimals, but will not replicate cells that reach only mitosis (union ofthe egg and sperm to formation of a reproductive cell) regardless of theprocess used. Production of the primordial or undifferentiated cells inboth plants and animals in a weightless environment can produce the sametissues during mitosis present in the original union of egg and sperm inanimals or plants, and possibly the extraction of apical meristemresulting in the germination of a seed in zero gravity and use of theundifferentiated parenchyma cells.

Current procedures cannot reproduce progeny of the originally unitedsingle cell at mitosis. There is a need to develop a process ofmanufacturing of the primordial cells also known as undifferentiatedcells into a source of germ plasma for plants or stem cells for animals(including man). The force of gravity has prevented the establishment ofa process that would allow the primordial tissue for replication of bothplants and animals in a more efficient and productive manner. Currentlythe only source of cells at the telophase stage of mitosis is theharvesting of fetus and associated tissues, including the cord tissueand blood from miscarriages obtained from hospitals, clinics and otherfacilities.

A proposed method is presented herein for reproduction of the cells inmitosis in both multi-celled plants and animals in a weightlessenvironment. The methods used for the reproduction of the cells at thetelophase stage of mitosis will be adjusted as necessary by personsskilled in the art, but taken from current technology with modificationsto adjust for a weightless condition or reduced gravity, includingpossible simplification of the process. Gravitational forces may governthe reproduction of animals in a weightless condition, but this fact hasprovided the opportunity for the replication of cells during mitosis isweightlessness.

The scientific literature describes the differences in the success inthe reproductive rate, and more specifically, the longevity ofindividuals that live at sea level vs. the tallest mountains (note: thecriteria is longevity, not mortality). The data only suggests thatgravity may be a factor in the reproduction of the human species. Agreater success rate for reproduction of the human species is indicatedby a greater gravitational force. An overlay of the greatest populationcenters coincides with those regions with the highest gravity. Gravityon Mars may present a problem for reproduction of the human species aswell since the humanoid is not adapted to the reduced gravitationalforce.

The understanding of cell reproduction in zero gravity provides afurther benefit beyond the union of “egg and sperm” in animals and theunion of pollen (or equivalent) with a plant ovary. The final stage ortelophase of mitosis occurs and replication of the undifferentiatedcells results in a population explosion of cells that can be processedand stored in suspended animation, such as freezing in liquid nitrogenor other low temperature environments or other techniques, to ensuresuspension of replication, for shipment to a gravitational force for useas totally undifferentiated stem cells in animals and undifferentiatedparenchyma (or like tissues) in plants.

The literature supports the premise that compromised” tissues exposed togravity at conception (produced as a result of gravity at conception)for plants and animals will not reproduce the parent tissue faithfullyand indicates serious concerns for space travel for long periods oftime. The literature and observations of long-term visitors to zerogravity provide such evidence.

The present invention is premised upon the theory that mitosis occurs incells from dedicated tissues (bone, muscle, etc.) that does not producethe cells of origin (bone, muscle, etc.) but will follow reproductioninto cells of origin that may be influenced by, but not in control ofthe parent cell (bone, muscle, etc.). Such cells are not the replicationof the parent tissue, nor new undifferentiated tissues, but a “K” cell(new terminology) that has no purpose, but can function. Such factorsmust be included to incorporate an artificial gravity technology thatmust be required for any long-term space travel or colonization of theMoon, Mars or any other body with a gravity force of less than one “G”.

Methods

Current practices on Earth for unification of egg and sperm in higherorder living biological systems (HOLBS) are commonly practiced in thereproduction of man and other animals. These same procedures are usefulfor impregnation of an egg with a sperm in a weightlessness environmentaccording to the present invention. All procedures used on Earth forimpregnation of egg and sperm in HOLBS, and other animals are useful forthe production of undifferentiated cells for the production of tissuesor for other purposes, such as reproduction of endangered species.

The procedures include, but are not limited to the use of a preservedegg and sperm held in a suspended animation phase that are revitalizedusing current techniques in a weightlessness environment and allowed tounite to form a single cell. The current techniques and advanced roboticprocessing are used to unify the sperm and egg to produce a cell capableof replication in the weightless environment described herein. Theunited egg and sperm preserved immediately upon the unification, canreplicate itself without forming a zygote that will develop into anembryo of differentiated cells and tissue, and can be utilized for thepurposes discussed (tissue reproduction, reproduction, etc.). The use ofthe procedure of using preserved egg and sperm of HOLBS and lower orderanimals in a weightlessness environment when and eggs and sperm aretransported to an on orbit facility, processed and unified inweightlessness, allowed to reproduce in a contained space, processedthrough a transitional production phase that may include, but is notlimited to and not necessarily in this order of the coating of theindividual cells, to prevent “grouping” (if necessary), gradualcool-down to suspended animation, chemical treatment (if necessary),into a vessel where the replicated cells can be stored in suspendedanimation. The replicated cells can then be transported back to Earthfor uses described.

Additionally, the replication and production as described herein for theunification of pollen (sperm) and ovary (egg) of the equivalency ofHOLBS and lower plant life in a state of weightlessness condition forthe purpose of production of undifferentiated cells is described herein.The procedure includes, but is not limited to routine isolation of thepyramid cells (undifferentiated tissues) in the apical meristem andculturing the tissues. However, the weightlessness environment willprovide an exponential growth of the undifferentiated tissues, yieldinghigh quantities of the cells that may be processed using a coating topreserve hydration and other life-sustaining parameters, a suspendedanimation process and packaging for transport back to Earth.

The principle is the same whether the cells being replicated forproduction are animal or plant cells. The cells being replicated are theresult of the male and female cell union from animals or plants. Thisunion can be accomplished in weightlessness with great success using thecurrent state of the art used on Earth. Alternatively, the union can beachieved by uniting a female and male cell on Earth under gravityconditions, placing the single cell in suspended animation prior to anycell division of the single formed cell, and transporting the storedcell to orbit or a similar environment for replicated production inweightless conditions.

The single cells produced by union of the female and male (egg and spermfrom animals; pollen and ovary from plants) can be replicated as singlecells in a production mode that will yield cells at an exponential rate,providing cells for use in tissues for body parts or branches or roots,depending on the progeny.

Plants

The present methods of reproduction of plants include cloning and seeds.Reproduction and use of apical cell reproduction has greatly increasedthe numbers of plants in a vegetative reproduction process. The processdepends on the isolation of the reproducing cells at the tip of a plantor plant part (root, branch, etc.) known as the meristem and successfulcloning of the limited number of cells at the undifferentiated stage ofdevelopment at the tip of the plant or other actively growing portionsof the plant (root, cambium, etc.). The process is efficient forherbaceous plants, but the “woody” plants cannot be reproduced in highnumbers because of the limited availability of apical cells. Cells inmitosis in quantity would speed the introduction of new varieties andspecies, including endangered species. The commercial advantage would besaving years of time between discovery and introduction of the plant tothe market.

The production of an unlimited number of the undifferentiated cells hasnot been possible because gravitation forces “pull” cell matter into“layers” (starch, etc.) that will start the processing of theundifferentiated cell into specific (differentiated) cells. The processin the presence of gravity allows the cells to differentiate into a stemor a root and guide the plant using gravity to form the stems, leaves,flowers, etc. that have been programmed over billions of years to formsuch structures given the “keys” that that are needed by the genes inthe genetic code. However, the plant will perform “naturally” at onlyone “G” or more to another limit.

Forces exerted by artificial gravity induced have been demonstrated by afew zero gravity experiments where plants were subjected to a “spinning”in the experiment to produce an artificial gravity. The experimentdemonstrated that such means of plant production cannot be producedusing limited experimentation. The plants were deformed, due to themigration of molecules within the cells (starches). The plant wasconditioned to formation of the root vs. stem, etc. in a one “G”situation, the only sequence the plant was acclimated to over a fewmillion years. The plants were also subject to the effects of gravityprior to being sent to zero gravity, and the effect of the “G” factor onthe plants may have also contributed to the failure of the experiment.Otherwise designed with the germination of the seeds in a spinningdevice that would mimic one “G” may have produced a different result,and healthy plants may be produced using this technique.

The plants sent into zero gravity had already been compromised as aresult of the initial introduction to gravity after germination and theadditional force of the “G” forces from take-off of the mission and hadalready programmed genetically the plant to produce roots in onedirection and stems in another. But it is possible to replicate andproduce undifferentiated parenchyma resulting in the unification ofpollen (sperm) and egg (ovary) in plants that are unified on Earth,preserved prior to any division of the united single cell, andtransported immediately to orbit for the purpose of producingundifferentiated cells capable of replicating identical cells forproduction of tissues used for parts of plants, and the plant itself,including, but not limited to stems, roots, flowers, seeds, fruits, andother tissues. Such tissues are preserved in a suspended animation statefrom orbit to use in the field using the current technologies nowpresent on Earth.

The present invention is premised upon the theory that a union of pollenand ovary (egg) in zero gravity will produce a cell that will go tomitosis and then reproduce that cell continually en masse or until agenetic break down in the cell(s) may occur that would disrupt theexponential reproduction of the same mitosis. The newly formed cells canbe harvested through an exudation process and stored (includingsuspended animation) until the cells are shipped back to Earth or someother base camp with sufficient gravity. The cells can then beincorporated using current techniques for use of undifferentiatedtissues. The apical cells of plants produced in orbit that reproducecells in mitosis that will faithfully reproduce the identical cells canbe used for multiple purposes.

The current practice for the production of plants by uniting the pollenand ovary on Earth is time-consuming and difficult, but using theundifferentiated growing cells produced in a weightless environment canproduce the same results with exponential numbers in comparison. Newgrowth from plant cells and tissue in weightlessness can provide asource of the undifferentiated cells for the manufacture of such cellsin an exponential fashion using the techniques described and/or othertechnologies that exist or to be discovered.

Once produced in a zero gravity environment, the undifferentiated cellsat mitosis can be stored and conditioned fora storage, and preparationsfor distribution of the manufactured cells organized for transportationto gravity and utilized as stock for plants. Undifferentiated planttissue en masse could be used in the production of fruit tree scions onstock already established. For example, the insertion of theundifferentiated cells from several varieties for a compatible nature(example: Rose Family—apples, pear, peaches, plums, etc.) can bestrategically placed on a sturdy stock (Rose Family). It would bepossible to reproduce several varieties of fruit, all strategicallyplaced on that stock to allow a “checker-board square (all fruit of acertain variety) facing along lines for harvest. The farmer wouldharvest the crop of plants facing into a row on both sides of the trees.The next maturing crop would be on another ninety-degree directiondepending on the sun and shade requirements of each species on the rootstock. This would allow the production of at least four varieties offruit from the rose Family to be harvested depending on the maturationof the fruit. Apples, pears, peaches, plums, etc. All have differentmaturation times and the same stock used to impregnate each of thespecies would make that rootstock more productive. The same care(fertilization and pest control) would only be given to one plant whileproducing four different Species, varieties etc., from the same plant.As described several varieties from the same species may be harvestedfrom the same root stock if maturity of the varieties will not interferewith pollination.

The same process can be used for stocks from other fruit or nut bearingFamilies and species. The production of these varieties en masse at analmost exponential rate would provide an industry with a prolongedseason with the same care of only the standard number of plants. Itwould be advantageous to back yard gardeners to have a season-longproduction of fruit from the same tree.

The technique for introduction of the undifferentiated parenchyma fromeach individual stock would be less time consuming than the presentmethod of introducing an already vegetative active branch from anexisting plant. The method will be to make an incision on the sturdystock in each of the areas where the specific variety will be positionedand introduce the undifferentiated cells with the necessary nutrient mixand patch.

Most plants will reproduce new growth if a limb is severed. There isreproduction of parenchyma cells at or near the severed limb ant theundifferentiated cells reproduce a new limb with all the characteristicsof the missing limb. The new limb will follow all the rules set by thatplant for a gravity environment, including the production ofundifferentiated cells spontaneously in normal gravity when new growthor trauma occurs. New undifferentiated cells can be introduced at suchsites or be artificially induced to provide the same results.

If the union of pollen and ovary for a plant occur in a weightlessstate, the plant would not have any instructions to reproduce anythingbut undifferentiated cells (parenchyma). The use of apicalundifferentiated cells may produce the same results in regard toexponential growth of cells for purposes of harvesting undifferentiatedtissues and that tissue can be replicated at an almost exponential rate.

Specific varieties can be selected for the use as scions. The amount ofa new variety produced in zero gravity can be enormous, and provide aload-time of the introduction of a new variety by years. The use of thistechnology for plants, especially woody plants with a long reproductivecycle could be tremendous considering the long time frame betweendiscovery of a new variety, etc. of a plant and introduction to theindustry. This is especially important in the efforts to save endangeredplants or when a need for a massive number a specific species isrequired for the detoxification of a region (radiation, etc.) isrequired.

The time between the discovery of a new and remarkable variety of aplant and the market place for plants can vary based on the life cycleof the plant. There are exceptions such as herbaceous plants liketomatoes that can be reproduced by seed and self-pollinated usingstandard processes without too much of a problem. However, woody plantsthat have life cycles that may be years long cannot be reproduced in anynumbers and consequently the timeline for introduction into the marketplace may be years after discovery. When a plant that has a uniqueproperty (including endangered species) and has a reproductive cyclethat may be more than a few years the use of zero gravity becomeevident. The seed or the use of pollen with an ovary may be an optimumvehicle in spite of being exposed to gravity during formation and needsto be studied because any material transported to zero gravity may havealready been exposed to the elements that will prevent cell reproductionof the primordial cells in bulk. The undifferentiated cells from agrowing plant placed in a weightless environment may be one of thesources of undifferentiated cells that can reproduce in an exponentialfashion. The cells in the primordial tissues or undifferentiatedparenchyma of apical meristems can be redirected to the replication ofthe undifferentiated cells in a manufacturing process.

Animals

The current method of harvesting stem cells for a useful purpose inanimals is complex. The use of all relatively non-differentiated tissuesfrom after birth has become the only source of “stem cells” under thatdefinition. It is known that even such tissues have merits. Problemsinvolving the “harvesting” of the placenta and related tissues obtainedfrom humans and distribution of such “refined” tissues using the currentstate of the art have become a political issue because the stem cellsharvested are programmed in a genetic sense to become zygotes (newbeings).

All mitosis in an Earth-bound reproduction will lead to reproduction ofcells leading to a zygote and beyond. Current methods of obtaining stemcells include using the unborn, the placenta, and forcing a furtherreproduction of such cells. This procedure usually ends with thestraining of “stem” cells in a selective method based on the molecularsize of a zygote versus a more advanced cell that can be eliminatedbased on size produced in a gravity environment. There are no realquality assurances with such a procedure. Reproduction of pureundifferentiated stem cells on a commercial basis is not available in agravity-based system.

There is an ethical question regarding a “formed” animal zygote versusthe use of undifferentiated cells that cannot reproduce into an animalunless there is an intervention that would require extraordinary stepsto produce an animal. The cells produced in a weightless environmentwill not be used for reproduction (although theoretically possible)during this process and will be manufactured for the purpose of saving,preserving and healing animals (including man). The cells will be singlecells without any direction on production of an individual or even aspecific organ. Undifferentiated tissues re-construct the cells inplants and animals continuously until death. The use of pureundifferentiated cells for the purpose of healing tissues is a gift foranimals, including man. Creation of the “perfect” animal is not anobjective, but should be carefully monitored.

The method of reproduction of animal cells at mitosis will be in regardto current practices. The process must be modified to take into accountthat the cells will be reproducing in a three-dimensional environment.Current practice only allows for a two dimensional expansion. Thecontainer for the expansion of the cells produced, storage and othermanufacturing concerns will be addressed. Cells that reach telophase ofmitosis will continue to expand in numbers (exponential) and can beconfined, exuded through a special process, preserved (includingsuspended animation) and packaged for distribution in an efficientmanner through the use of robotics. The present method utilizes thecurrent technology for the extraction and reproduction to provide amechanized system for the manufacture of primordial or undifferentiatedtissues from both plants and animals (including humans) in zero gravity.

The present invention also encompasses a method of producing stem cellscomprising obtaining purified stem cells from an embryo of new-bornanimal and culturing said stem cells in a weightless condition orenvironment resulting in the cell replication of identical stem cells.Further included in the present invention is a method of producingundifferentiated cells from an animal, such as cells from liver, kidney,heart, skin, and other cells from the animal body including organs forthe purpose of replication of undifferentiated cells including obtainingthe undifferentiated cell from the animal, and culturing theundifferentiated cell in a weightless condition or environment resultingin the cell replication of identical undifferentiated cells.Additionally, the undifferentiated cells may be a subcutaneous skin cellobtained by harvested from the animal, wherein the culturing results innear exponential replication the cells overtime.

Further, the present invention encompasses a plant or animal orundifferentiated cell thereof produced by any of the methods describedherein and in any of the priority documents, wherein said plant, animalor undifferentiated cell thereof comprises at least one identified genethat has a change in expression level as compared to the gene expressionprofile of control cell, wherein the identified genes are associatedwith adaptation to the hostile environment.

More specifically, the egg and sperm are united using standard in vitrofertilization (IVF) techniques for harvesting human or animal eggs,collecting sperm and inseminating the egg with the sperm in a laboratorydish in IVF culture medium. The dish is then placed in an incubator at acontrolled temperature which should be the same temperature as thefemale species' body. It generally takes 18 hours for fertilization ofthe egg to be complete.

If the fertilization or the union of the egg and sperm occurs inweightless conditions then the single fertilized cell can be maintainedin the incubator with the change of medium over time. If thefertilization occurs in a gravity condition or environment, then afterfertilization is complete, and before the fertilized egg or zygotedivides, the fertilized egg should be immediately transferred to aweightless condition for culturing. However, if there will be a delayuntil the culturing in weightless conditions can occur, the fertilizedegg should be placed in suspended animation before it divides into twocells, such as freezing the egg. At a later time, when the fertilizedegg or zygote can be cultured in a weightless environment, it should berevitalized from its suspended animation, and cultured under theconditions to expand the fertilized egg into a culture ofundifferentiated stem cell using known stem cell culture techniques thata publicly available in scientific publications, patents and bydisclosed on-line methods and media.

For example, a medium useful for the isolation of embryonic stem cellsis “ES medium.” ES medium consists of 80% Dulbecco's modified Eagle'smedium (DMEM; no pyruvate, high glucose formulation, (InVitrogen orSignma), with 20% fetal bovine serum (FBS; Hyclone), 0.1 mM13-mercaptoethanol (Sigma), 1% non-essential amino acid stock (Sigma orother known sources). Preferably, fetal bovine serum batches arecompared by testing clonal plating efficiency of a low passage mouse EScell line. FBS batches must be compared because it has been found thatbatches vary dramatically in their ability to support embryonic cellgrowth, but any other method of assaying the competence of FBS batchesfor support of embryonic cells will work as an alternative. But anyknown media for culturing the replicating stem cells can be used andtested by the scientists performed these experiments to select theappropriate medium to obtain optimum results. Appropriate plant cellculture media known to skilled persons can be selected to cultureundifferentiated plant cells according to the present invention.

The cells are cultured in 3-dimensions by simply suspending the cells ina closed culture vessel in the weightless environment which will keepthe cells suspended without the need for any agitation as the cell willnot settle to the bottom of the vessel that they would in a gravityenvironment. Any know methods of 3-dimensional cell culture can be usedto culture the replicating undifferentiated stem cells, which couldinclude culturing methods from Mina Bissell's laboratory, such as forexample disclosed in J. Cell. Sci., 2003 June 15; 116(Pt 12):2377-88.

Somatic embryogenesis has been the model for mass clonal propagation ofa diverse array of higher plants, and also a model for studies of embryodevelopment and cell differentiation. For certain species,differentiation into somatic embryo requires cells to undergo atransitional stage, whereby embryo-like structures are formed. Theseembryo-like structures are comprised mainly of undifferentiatedparenchyma cells. The conversion of these structures into embryos isregulated by a number of different genes under normal conditions.However, when submitted to microgravity conditions, the cells inembryo-like structures may behave differently. The principal objectiveof the experiments of the present invention is to evaluate whether cellsof a monocotyledonous and a dicotyledonous plant develop normally fromthe transitional stage into normal somatic embryos under space(microgravity) conditions. Histological (structural) and geneticanalyses will be performed to assess the cells.

Additionally, suspension cultures have been widely used for tissueculture and mass clonal propagation of a diverse array of higher plants,and also as models for studies of cell development and differentiation.Analysis of these cells will verify structural and genetic changes inplant cells submitted to the effects of microgravity. In addition, cellgrowth and replication will be assessed visually. Structural changeswill be performed through histological analyses, including lightmicroscopy, transmition electron microscopy (TEM), and if feasible,scanning electron microscopy (SEM). Genetic analyses will be performedto evaluate differential gene expression under microgravity.

MATERIAL AND METHODS Plant Material

Monocotyledonous model species: Encyclia plicata, an endangered orchid

Dicotyledonous model species: Arabidopsis thaliana, a small floweringplant that is widely used as a model organism in plant biology and itsgenome has been completely sequenced, providing extensive genetic andphysical maps

-   -   Dicotyledonous model species: Caesalpinia puicherrima, a        tropical flowering tree.

Dicotyledonous model species: Tabebuia aurea, a tropical flowering tree.

Ground Cell Suspension

Cell suspension cultures are initiated for all species above mentioned,using callus tissue. Callus is comprised of undifferentiated parenchymacells, with no pre-determined growth pattern.

For the orchid suspension cultures, the MS culture medium (Murashige andSkoog, 1962), modified with 1 mg/L 2,4-D (2,4-dichlorophenoxic aceticacid), an auxin, and 0.25 mg/L 6-BA (benzyladenine), a cytokinin isutilized. Cultures are multiplied and built up in 125-ml Erlenmeyerflasks, under agitation in an orbital shaker at 109 RPMs. Arabidopsisand tree c ell suspensions are cultured on MS medium modified with 1mg/L 2,4-D. Once a significant amount of cells are produced, they aretransferred to 10-ml opticells. Also WPM (woody plant culture medium)medium (Lloyd and McCown, 1986) modified as the MS medium above may beused for woody species of plant.

OptiCell™ is a sterile, sealed cell culture environment between twooptically clear gas-permeable growth surfaces in a standard microliterplate-sized plastic frame with specially designed ports for access tothe contents. OptiCell allows an ideal environment for cell growth,microscopy, treatment, selection, separation, harvest, storage, andshipping. Optically clear gas-permeable growth surfaces allow diffusionof oxygen and carbon dioxide for optimal cell growth and permitmicroscopic examination at any stage of any cell process. OptiCell iscompatible for use with standard, phase contrast, confocal, andhigh-resolution time-lapse video microscopes and takes up a fraction ofthe space of conventional cell culture devices. Access ports allowaseptic access to the interior and its contents.

Space Hardware

Each opticell contains about 10-12 ml of cell suspension. Opticells aremaintained in quiescent conditions for both ground and spaceenvironments and are evaluated periodically through visual observationsfor cell growth and development. Under microgravity (space) conditions,opticells are arranged in a C-Hab hardware developed by Bioserve,University of Colorado, comprised of 6 individual Opticell cell culturesystems, peristaltic pumps and a control circuit board. The C-Flabhardware allows the transfer of 1 ml of suspension from one opticell tothe next during transfer of cells to fresh medium. An aluminum base andan extruded aluminum outer box with a clear optical window provide thesecond level of containment. Visual evaluations in space are performedwith the aid of video cameras. The C-Hab is associated with CSI cameramodules. Each of the camera modules contains up to three analog colorvideo cameras, fitted with either microscope adaptors or standard lensesfor macroscopic view. This allows the observation of cell growth andreplication throughout the period of experimentation in space. Stillimages (jpegs) are fed to the ground periodically during the entireperiod of the experiment, thus generating a time lapse for cell growthand replication. The hardware and related control software are testedand evaluated previous to launch.

Evaluations of Cell Suspensions

Samples from suspension cells maintained in opticells, under both groundand space conditions are collected and compared for histological andgenetic analysis: [0141] 1. Histological analyses: Cell suspensions areprepared for light and electron microscopy. Opticells are compatible foruse with standard, phase contrast, confocal, and high-resolutiontime-lapse video microscopes. Cells are examined microscopically oneither optical growth surface or in between. Oil immersion lenses (up to100x) are used on the membrane without disruption or contamination. Themembrane is sectioned for small scale staining and microscopy.Additional samples are removed and fixed in Glutaraldehyde forsubsequent evaluation of cell ultrastructure through TEM and SEM. Theseanalyses are performed for all species. [0142] 2. Genetic analysis: Geneexpression analyses is performed to evaluate possible genes that areeither turned on and/or off under microgravity. Suspension culturesmaintained in space are fixed in RNAlater (Ambion) liquid preservativethrough the Kennedy space center fixation tube (UT), hardware designedto provide proper containment of fixatives for biological samples inspace placed inside the C-hab environment. RNA is isolated and comparedfor both ground and space suspension cultures to evaluate geneexpression. Molecular biology techniques for reverse transcriptasepolymerase chain reaction (RT-PCR) and/or copy-DNA amplified fragmentlength polymorphism (cDNA-AFLP) and gel electrophoresis according tostandard known techniques are used for gene expression analyses inground (TREC lab). Microarray analysis of gene expression is performed.[0143] 3. Additional evaluations: Preliminary experiments in groundenvironment (on earth as opposed to in space) are useful to addressdetails and to generate practical solutions for the successfulestablishment of experiments in space. Evaluations for both earth andspace experiments provide additional information and data specific forthe Opticell cell culture system environment and include specificconditions for the opticell cell culture system environment, such ascell growth rates, cell densities, subculture frequency, and size andcondition (live vs. fixed) of samples for histological and geneticanalyses.

Experimental Design and Statistical Analyses

Controls are comprised of 10 ml of suspension cultures maintained in 6opticells under ground-laboratory conditions. Treatments are comprisedof 10 ml of suspension cultures maintained in 6 opticells inmicrogravity. The C-Hab environment has been accommodated into modularracks inside modules provided by Spacehab, placed in the Space ShuttleEndeavour, launched on Aug. 8, 2007, Mission STS-118. The experiment ranfora period of three months approximately, whereas the controls aremaintained in ground and the treatments are maintained in theInternational Space Station (ISS). Treatments were returned to groundthrough another space shuttle mission on Nov. 8, 2007. Analysis ofVariance (ANOVA) are performed using SAS statistical package foranalyses of results. Means for treatments will be compared for allparameters evaluated using the Tukey's test.

Data were collected regarding plant growth characteristics and theresults of these studies showed that the cells grown in the weightlessenvironment on the ISS replicated to a greater mass than the controlcells grown in a laboratory under the earth's gravity with all otherculture conditions the same. One of the two tree species demonstratedsignificant growth on orbit in the weightless environment as compared tothe ground controls.

The studies support that cells would replicate rather than duplicate andthat exponential growth of the cells will occur as long as theundifferentiated cells are nurtured and maintained in a weightlessenvironment. The results show that that cell growth on the ISS in threeof the species of plants demonstrated higher growth than the controls onEarth. The third experiment (Moncot—orchid species) was contaminatedwith a yeast infestation and is being examined to determine the speciesand information regarding gene expression and replication.

Effect of Weightless on Growth of Replacement Cells in VisitorsMaintained in a Weightless Environment

As noted above, there have been reported studies of humans who lived ina weightless environment, such as on the Space Station, that show thatthese individuals lost body mass during their time in this weightlessenvironment even though these individuals exercised during their stay inthis weightless environment. In a weightless environment, evenestablished cells produce undifferentiated cells immediately adjacent tothe existing cells that “mimic” the cell, but do not perform properlyand do not produce the tissue of origin (bone, brain). The tissues thathave the greatest amount of circulation will be effected first, such asblood and brain, and these tissues will be compromised. All long termorbital and interplanetary missions should take under consideration theloss of body mass regardless of the precautions incorporated into aspace mission, including exercise, supplements and the like to maintainbody mass. The rule concerning exercise in a weightless condition may beharmful because of the degradation of the body and replacement of thedestroyed cells by a cell that will not be a replicate of the originalcell. The cell reproduced in normal cell reproduction in one “G” doesnot have the influence of the one “G” and replicates into a “K” cellwhich functions, but is not the replicate needed for normal function inthe tissue. For example, a new cell in a muscle tissue should functionas a normal muscle cell but it does not.

All long-term missions should be reconsidered until there is anevaluation of this concept and examination of the information presentedherein regarding the formation of non-differentiated cells and unlimitedreproduction of such cells. Thus, the risk to individuals who spend anyextended time in a weightless environment is based on the premise thatundifferentiated tissues can reproduce as “pure” undifferentiated cellsproduced by a male and female union in weightlessness.

The effects of long-term exposure to weightless conditions is governedby the knowledge that the reproduction of existing cells in a visitorwill not be faithfully reproduced to provide replicates of thegenerating tissue. Not wishing to be bound by a specific theory, thepresent invention is based on the theory that the cells that continuallyare produced do not form into tissues (bone, muscle, blood), and willnot develop or mature into the differentiated cells from the originalcells. The cells will not differentiate properly into the parental cell,but under the weightless environment will form a cell not functional tothe specific task intended for the cell, such as bone, muscle or blood.The term “K” cell' is only a designation, but is being used herein todescribe a cell not yet studied, but present in all long-term spacevisitors.

In an effort to solve the loss of body mass in weightless environments,the experiment concerning the use of embryonic tissues in the “SouthAfrican” experiment confirmed the theory that the reproduction of newcells does not follow a normal growth pattern. The tissues derived fromanimals within hours of lift-off in Russia provided informationconcerning the techniques that may be used in future missions. The SouthAfrican mission did provide information including the reproduction oftissues in a weightless environment NOT reproduced with a sperm and eggin zero gravity, or a “new” seed or egg of a plant recently pollinated.The expansion of cell growth did not take on the normal pattern andinstead became an unrecognized pattern. Again, the experiments provideinformation that lead to the confirmation of the theory that all cellsderived from tissues in weightless environment are not orientated toreproduce the same type of tissues adjacent to those tissues.

The cells reproduced in likely become “inert” cells meaning that thecells do not have the same specialized function as the adjacentdifferentiated cells in the tissue from which these cells originate andare reproduced. In other words, the longer an individual stays in space,the higher the probability that the body will degrade, and may do so ata higher rate in the individual who stays in a weightless state. Infact, such cells may be anaerobic in character and pose a future cancerrisk since the cells do not follow the “Krebs cycle,” resulting infermentation rather than an aerobic cycle.

Individuals in orbit weightlessness have been experiencing loss of boneand muscle mass, and other tissue. The areas of the body with the leastamount of circulation (bone, muscle, tendons, fat,) will experience themost trauma because of the circulation to those areas of the body.

Intense exercise in zero gravity has not been able to slow the loss oftissues from the body and may be counter-productive to long-term intactcell survival, since cells are created and destroyed at much higherrate. It is known that the body is reproducing new cells on a continuousbasis and the more exercise is performed, the more cells that becomeexposed to the reproductive process and the more undifferentiated cellsthat are produced. The reason for the loss of mass in the bone, muscleand other tissues is that the reproducing primordial (stem) cells thatdo not have a direction (bone vs. muscle, etc.) die or form a cell weare not familiar with unless we look. A lock at the space traveler in aweightless environment without the proper devises to allow for anartificial gravity may create a major problem.

Given that the human body continuously reproduces specificdifferentiated cells for each organ and that such cells may only beprimordial with no direction, there is a strong possibility that theprimordial or undifferentiated cells do not develop into thedifferentiated tissues intended (such as bone, muscle, or otherspecialized cells) and may degenerate or possibly form a cell that weare not familiar with in the reason of normal thinking. It is known thatthere is a loss of tissue and all orbital and interplanetary visitorsshould be examined for necrotic or other cells in the areas of the bodythat have low growth, including the brain and other organs studied.

Many technologies have been applied, including vigorous exercise, toslow or prevent this loss of tissue. The latter may in fact be harmful,since exercise promotes the life and death cycle of cells, and death ofthe cells does occur, but the new cells produced are undifferentiatedwith no program to produce the differentiated cells which they are toreplace. Even the new reproductive tissues of visitors becomeundifferentiated when produced and either transform into the tissueitself (with a “lag” time) into the bone, etc. or die or become an“inert” tissue with no specialized function.

On-Orbit Manufacturing Facilities for Stem Cell Production

The use of an on-orbit manufacturing facility or “wheel” in space usingnew technology will allow production of primordial or undifferentiatedcells and tissues from plants and animals and provides an environmentfor those that harvest the cell crop (FIG. 1).

The reproduction of the original cell obtained from the union of thesperm and egg in animals or the pollen and ovary in plants occurs in a3-dimensional fashion in a culture vessel or container, since no gravityis involved, the cells will replicate and remain in suspension floatingin the media in the culture vessel. The cells will continue expanding inthe culture vessel until the “yoke” of the process is expended until thesize of the cell population has reached its maximum capacity in thecontainer. Then, the newly formed cells can be transferred or exudedfrom the culture vessel into a container that will provide a suspendedanimation system for storage and shipment to Earth. A system will bedescribed for the manufacture of undifferentiated tissues in aweightless site, and the cells will be cultured in the weightlesschamber in the on-orbit manufacturing facility shown in FIG. 1 or in asimilar type chamber in another space vessel or station.

Manufacturing

Capturing the reproducing cells from plant and animal primordial tissueswill be a mechanized procedure. Cells (individual or two or more) can becaptured and pulled through a mechanism to encapsulate the cell(s) in asheath that mimics the cells walls of single celled organisms such asbacteria. The storage of the undifferentiated encapsulated cells must bestored in a weightless system where the metabolism and physiology of thecells proceeds into a state of suspended animation. Temperatures andstorage conditions can be adjusted to provide an optimal condition thatallows the harvested cells to be stored. Slam freezing is used topreserve semen specimens for cattle now and can be used. The process ofthe cells must be slowed to a level that will allow storage and eventualhandling of the cells when returned to a gravity environment.

Yeast may represent a model for replication of both plant and animalundifferentiated tissues since yeast provides the preparation(substrate), innoculum (undifferentiated cells and/or tissues),cultivation (providing ideal conditions), harvesting (separation ofcells from the process), cooling and storage of the harvest attemperatures not to interfere with gravity.

The aerobic respiration (presence of O2) expected in the culturingprocesses with both plants and animals will be interfered with byanerobic respiration (lack of O2) due to the containment of theproduction and the difficulty of feeding the entire nutrient system withadequate oxygen, especially near the end of the exponential growth phaseof the manufacturing process. Aerobic respiration has both the substratelevel and oxidative phosphorylation while anerobic respiration has onlysubstrate phosphorylation. Both use hexose as a fuel (glycolysis). Aproblem in the manufacture of the undifferentiated cells is that duringthe anerobic respiration expected in the process, the final electronacceptor through alcohol will be other organic molecules such aspyruvate, acetaldehyde, etc. that will be toxic to the total mass ofundifferentiated cells. In the aerobic respiration, the final acceptoris oxygen and presents no problems.

Respiration resulting from undifferentiated cell replication occurs inboth plant and animal cell cultures. The exponential expansion of cellsfollows the general guidelines for plants and animals:

-   -   1. Sugars (Hexose in plants) is the fuel.    -   2. Zymase in plants is an oxidizing/reduction enzyme to act as a        catalytic agent to covert sugar into labile intermediate        products.    -   3. Animal conversion during the metabolic process follow the        same general format (Kreb's Cycle).

Plant Model

The expansion of the cells will produce by-products depending on theoxygenation of the system but in containment, both aerobic and anerobicrespiration will take place at the same time, producing toxicsubstances:

The amount of byproducts accumulates and alcohol with aldehydes, etc.requires removal from the manufacturing system through the use offilters.

The production of undifferentiated cells requires the filtering of thetoxic byproducts and the injection of oxygen into the system. Theproduction of cells requires careful consideration of the “criticalmass” determined by the maximum numbers of cells that can be produced bythe available nutrients, the supplied oxygen and the removal of wastes.A “flow” system is highly probably where a reaction chamber allows a mixof the nutrient, “seed” undifferentiated cells and oxygen can produce acontinual flow of the nurtured “seed” cells with a proper oxygenenvironment would expand into a container vessel that has environmentalcontrols. However a more simplistic model is useful that can bewell-adapted to a robotic system, for example, such as disclosed in FIG.2. The configuration of the system and its manufacture for eachcontainer is based on the following parameters:

-   -   1. Volume of reactor    -   2. Amount of substrate (sugars).    -   3. Amount of innoculum required.    -   4. Filter system for toxic byproducts    -   5. Injection of Oxygen    -   6. System for cooling reaction during progression of reaction        and cooling for storage

If the efficiency of the “Volatiles Filter” proves to be inefficient, aseparator may be necessary in the system. The manufacturing unit ispreferably designed toward robotic manufacturing with the followingconsiderations:

-   -   1. The manufacturing unit is at a pre-determined size to contain        the initial substrate and also serve as the storage unit for the        processed cells return to Earth.    -   2. The substrate environment is carefully monitored for        parameters that will insure a proper environment for the        injection of undifferentiated cells (or tissues) into the        substrate.    -   3. An oxygen injection system that provides levels of oxygen to        keep the process aerobic and evenly distributed within the        nutrient.    -   4. A re-circulation system with a exit into a filtering system        for removal of toxic volatiles also assists in the distribution        of oxygen. The system is preferably designed to prevent any high        movements of nutrients and cells to maintain a “non-gravity”        condition.    -   5. The exponential expansion of the undifferentiated tissues        will cease because of the amount of nutrient, size of the        container and/or    -   6. The toxic materials that may result.    -   7. The unit can then be reduced in temperature to below the        level required for respiration and shipped back to Earth.

The payload of undifferentiated cells will be retrieved much like theGet-Away-Special Program in the 1980's and the SpaceHab system andreplated with another “factory” Such factories can replicate theprimordial or undifferentiated cells and tissues of both plant andanimal on a commercial scale. The advancement in technology will allowfor better efficiency with each mission.

Production

The primary cells used to replicate identical cells in weightlessnesswill produce cells in an undifferentiated state much like a “seed” incrystallization in minerals or super-saturated solutions. Biologicalprocesses may follow different patterns, but the replication of the seedcell will produce undifferentiated cells in an exponential fashion.Therefore, there is a need for a device to control the replicated “seed”primordial tissues to produce encapsulated cells.

Biological “factories” such a female termites can be used as model forthe replication in zero gravity. The primordial tissue in an exponentialexplosion needs to be controlled as with the queen termite. The processinvolves the direction of the undifferentiated tissues through aprocessing chamber where a protective coat is applied. The process wouldallow a continuous flow of the primordial cells into the encapsulationprocess. The cells would then be directed into a chamber that isdesigned to provide a conditioning process for processing the cells intoa state of suspended animation. The processing of the undifferentiatedcells can be conducted using current technology with modification knownto persons skilled in the art of cell culture and manipulation. Use ofultra-light components of a non-metallic nature may be essential toprovide not only a gravity force, but also an electro-magneticinfluence. Containment of the production in a shielded environment toprotect the production and maintain the sterility of the cultures willbe necessary.

Selection of the egg and sperm is of great importance. The animals mustbe selected to provide the egg and sperm at an early age. The break downof the genetic code in latter years is well known for both egg and spermEven the generation prior to the donors must be considered. Thetelophase of mitosis of the union of the egg and sperm will provide stemcells for the individuals in a particular blood type and other tissuematching characteristics to match the donor with potential animals oflike character. Banks of eggs and sperm in the case of animals(including man) and pollen, plant female organs and undifferentiatedcells, such as parenchyma can be stored in suspended animation forfuture generations. It is expected that individuals will “bank” spermsand eggs in the early years of their sexual years to insure thecapability of regenerating or even replacing body parts. The bank ofsperm and eggs can be held in suspended animation. If a situation ariseswhere there is a future need for stem cells, the “pure” stem cells willbe available.

The present invention comprises the methods and facilities so that purestem cells can be reproduced in a factory mode. The cells will onlyproceed to mitosis and will not produce undifferentiated cells so thatno embryo will develop from the original single cell. The union of asperm and egg in zero gravity will not result in the production of azygote capable of developing into an embryo and then into a normal lifeform. This premise is based on the observations of maladies that occurto space visitors from both the US and Russia who spend some time in aweightless condition. The present invention is based on the theory thatthe undifferentiated cells produced by specific tissues (bone, muscle)did not differentiate into the intended differentiated, specializedtissues (bone, muscle) and became necrotic (Reference). The additionalnon-productive cells produced in a weightless condition did not have the“gravity” message required to reproduce in a gravity environment and areimbedded in the normal tissue and interfere with the “normal” cellssurrounding the tissue.

The additional data provided by the South African space attempts alsolead to a conclusion that primordial cells (undifferentiated) can andwill reproduce in a 3-dimensional fashion. The cells produced were theresult of already compromised (by gravity) cells that did indeedreproduce in the 3-dimensional environment. However, the instructions onreproduction (genes) were not only compromised by the union of egg andsperm under gravity, but also lift-off that provided many more gravityforces that certainly migrated the starches, etc. and effected theexperiment. The production of undifferentiated cells in volume can beachieved using the present technology in a weightless condition.

Design and Function of a Space Craft Using a “Clutch” Technology

The current visitors to space in orbit and destined for other planetsare subjected to a weightless environment where extensive documentationpoints toward degradation of the human body. Long space voyages to Mars,for example would not be practical and even suicidal. A method ofproviding an artificial gravity has been under study for decades, butdocking of a space craft, conducting experiments in weightlessness, andstill providing an environment where a simulation of gravity are presenthave not been solved until now.

The present invention comprises a space craft (as shown by the diagramin FIG. 1) that can be assembled in orbit using a “tinker toy” method ofconstruction using capacity figures for the space shuttle or moreadvanced vehicles. A puncture-proof inflatable two-piece (“clutch” andwheel) can be put in place around the stationary central weightlesschamber. The central “weightless” chamber can be used for docking andexperimentation. An adjacent outer segment (wheel within a wheel”) is a“Clutch” that will be at neutral or rotate with the outer wheel. Theneutral condition will always exist in inner “Hub”. The “clutch” can beslowed to “Neutral” and air-locked” to allow the transfer of personneland/or supplies into the “clutch”. The “clutch” system is sealed andbegins to rotate to catch up with the outer spinning wheel. Once it“catches” the speed of the outer rotating wheel, it can mate with thewheel and transfer of the personnel can take place. The rotation of the“clutch” allows for transfer from a neutral docking zone and laboratorythat allows experimentation in a weightless environment to the outerwheel that mimics Earth's gravity. The wheel may be approximately 100meters or more in diameter and include at least four to eight “spokes”.The vehicle can be “mated” to the existing space station.

The present invention further comprises an electromagnetic “track”system that will interface between the inner capsule (docking,laboratory, control area, and other compartments), the “clutch,” and theouter wheel to provide a friction-less interlace. A ball bearing systemcan be engaged during transfers between the “capsule” (weightlesscondition), the “clutch” and the wheel during transfer of goods andpersonnel and act as a back-up for the electromagnetic system. Therotation between the “Neutral (Capsule), the “Clutch” and the “Wheel”can all be interfaced with electromagnets that will allow for theproduction of electricity.

The present invention additionally comprises a computerized system ofweights and balances that are necessary to adjust the entire system.Transfers of personnel and equipment will require a weight and balanceto this relatively sensitive system and can be accomplished by pumpingfluids or movement of weights in the spokes of the “wheel” to counteractthe movement of supplies and personnel to the “rim” of the “wheel”.

The present invention also comprises the spokes of the outer wheel thatwill act as the elevators to the outer region of the wheel and toprovide ballast for balance of the system. The “rim” of the wheel willcirculate at a speed that creates a centripetal force that will mimicEarth's gravity and allow the inhabitants and plants requires forsubsistence to exist in a near-nor mal condition. The “rim” will requireadjustment by visitors, but there some individuals that do adapt to suchan environment and function. The sleeping “cocoons” can be arranged toallow for maximum benefit of the centripetal force. Plants can also bepropagated in such an environment.

The mathematics to calculate the circumference of the wheel to create anear Earth simulated gravity is well known. Examples are given in apublication “Artificial Gravity and the Architecture of OrbitalHabitats” by Theodore W. Hall, presented at the 1^(st) InternationalSymposium on Space Tourism, Bremen, Germany, 1997 No definitivecalculations will allow for a prediction of comfort in a space habitat,but the circumference of the wheel will be determined by the adaptationof individuals to the physical conditions and stamina in suchconditions. A selection process may be necessary to determine personnelfor such missions.

Providing an artificial environment that mimics gravity is alsonecessary based on review of the data and understanding the principalsof cell reproduction in weightlessness. A space craft traveling at aconstant velocity in interplanetary flight will create a weightlessenvironment and be subject to the same science already discussedconcerning orbital missions. A “wheel” and “Clutch” arrangement must beincorporated into any vehicle attempting a long interplanetary flight,including a voyage to Mars.

Spacecraft Structures and Electromagnetic Sub-System

The Earth is protected from the Sun's radiation and other particles bythe Earth's ionic shield. Mars and the Moon do not have such a field andis cited as a reason for the inability of these bodies to maintain lifeon our scale. The Earth's ionic shield is the result of the expulsion ofa positive charge from the Northern polar region that forms aelectromagnetic force that forms a spherical force field covering theplanet and terminating at the Southern polar region. The system thatprovides the electromagnetic shield is powered by the motion of thevarious metals (primarily Iron) in the heated core as described bycurrent science.

The present invention further comprises the space craft described hereinwhich will have a abundance of electrical power developed by theinterface of the “clutch” system using simple electromagnets. Theweightless inner portion of the hub must remain stable for the purposeof the docking of a visiting space craft, experiments, and otherfunctions. The “clutch” will also remain in a position adjacent to themain facility. However, the movement of the outer wheel will alwaysprovide a field production at the interface of the clutch and the wheel.The continuous electric power produced can be used to produce aelectromagnetic field based on the same principles that protect Earthfrom the damaging radiation, etc. from the Sun. The portion of the spacecraft providing the positive charge (as the North Pole) will be directedtoward the sun and a receiver will be placed at the opposite end of thespace craft. The positive charge may be on an extension boon to providea suitable profile for deflection of harmful radiation and other harmfuleffects from the Sun. As with Earth as a model, a proper profile for theamount of power required to achieve protection from harmful radiationcan be determined. The amount of power to accomplish a protective shieldwill be low because the power supply at the positive side will bereduced to a certain degree, but retrieved at the negative pole. Thereare no ground cables in space. The principles that protect the Earth areapplicable in space.

Acceleration of Adaptation and/or Evolution

When in a weightless and/or near-weightless environment, typically inspace and on-orbit, but also in any and all such equivalent non-weightand weightless circumstances, there exists cellular materials andwherein a process or processes are performed in those environments,specifically in the absence of a “weight-forcing condition” (acircumstance where weightlessness prevails), specific genetic attributeswill be altered, i.e., either switched on-from-off or switchedoff-from-on, and that certain of these gene characteristics may beidentified, as to their function and nature, and may consequently andpurposefully selected and altered in order to achieve specific,valuable, and useful outcomes, to derive targeted cellularcharacteristics and accordingly, behavior, performance, and function,and/or that result in the securing, development, and/or enhancement ofcertain preferable and valuable attributes of said cells, whether aspart of a cell, a cellular mass, a cellular volume, or a livingorganism, in whole or in part.

Wherein these cellular components and sub-components including cellularelements, genes, DNA, RNA, enzymes, hormones, etc., may be tiedindividually or collectively in structure, process, and function, suchthat they act to produce characteristics, attributes and capabilitiesboth cellular and when in-complex and mass as may be observed with aliving organism or organisms, such that they express living behaviorswhich include, but are not limited to increased or decreased ormodulated metabolic rate, i.e. respiration, transpiration, anabolic andcatabolic activity levels or both, up and/or down regulated, increased,decreased, altered or modulated nutrient uptake, increased, decreased,altered, or modulated waste and byproduct output, modification oralteration of water and fluid uptake or release, alteration ofstructure, i.e., modification, adaptation, or variation of cell wallthickness, structure, or composition, alternation of character asrelates to external environmental pH (acid/base) conditions, salinityand mineral presence, ionics and salts, water, temperature variations,both high and low extremes as well as cellular character, performanceand viability in the presence of rapid temperature excursions, orcombinations of external chemical, thermal, physical, energy,electromagnetic, radiation, solar, vibrational, acoustic, magnetic,paramagnetic, gravitational, weight-forcing conditions. In other words,all external conditions which may affect or interact with the cells,cellular mass or volumes, components thereof, or organisms in whole orin part may be modulated.

Also contemplated by this invention is the alteration of thosebiological and cellular processes, activities reactions, motions,behaviors, process induced reactions and behaviors and the like whichare deliberately conducted or affected or driven or occur in a“zero-weight-force” (so-called microgravity) environment. This includes,but is not limited to, the alteration of genes, DNA, RNA, enzymes,hormones, and all biologics known to those competent in the art ofbiology and cellular sciences, which result in the creation of and/ordevelopment of cellular components, cells, cellular masses and volumessuch that they or their complexed resulting cellular organisms, may bealtered, modified, and enhanced to yield valuable and useful newcellular types and variant organisms including, but not limited to, cowsresistant to mastoid staphylococcus disease, cattle resistant to mad cowdisease, wheat resistant to wheat rust, soybean crops resistant tosoybean rust, agriculture crops resistant to ralstonia, citrus capableof growing in Northern latitudes, Jatropha, Camelina, and other thirdgeneration biofuel crops capable of being grown in the Continental USlatitudes, citrus resistant to canker and greening, interbreeding ofTamarix to overgrown existing invasives in the Western US to eliminatewater losses, tailored halophyte production and growth, minimization ofwaterway invasives through retailoring of hydrilla, water hyacinth andegeria species, modification of grapevines to eliminate grape blight,modification of corn, soy, wheat, rye, rice and multiple other types offood crops enabling them to be grown and viable in drought conditions,in harsh wind and climate environments, or in adverse soils and a widevariety of other biological and cellular and organism modifications,alterations, and restructurings of similar usefulness and value andnature.

The present invention recognizes the value of the accelerated andsustained proliferation or replication of undifferentiated cells fromplants and animals in weightless conditions as an opportunity to selectfor organisms adapted to specific, even harsh, environments. By exposingthe proliferating or replicating undifferentiated cells to one or more“non-natural” environmental conditions while in orbit (orweightlessness), one can “force” the cells to express genes that enablethe cells to adapt and survive in these abnormal environments. Theselected cells can be cultivated to develop into an organism that wouldbe adapted to the specific environmental conditions to which itsprimordial cells were exposed.

The present invention comprises “replication processes” that occur in aweightless environment as contrasted to the normally occurringreproductive processes that result in progress or development throughnormal maturation stages and cycles resulting in the differentiation ofcells which are known as “reproductive processes” on Earth. However, onorbit in weightless or microgravity conditions, cellular activitiesoccur and are referred to as “replication” which can also be referred toas “duplication” or “proliferation” of copies of cells that areidentical in structure and function to their originating predecessors.This process is known as replication and results in an undifferentiatedcell replicating or duplicating itself without differentiating into amore specialized cell with a predetermined function. Thus, the processallows for the production of large amounts of undifferentiated plant andanimal cells in the weightless or microgravity environment.

As used herein, the term “undifferentiated” means a primordial state ofa cell or cells capable of differentiation and proliferation to produceprogeny cells that can be physiologically, biochemically,morphologically, anatomically, immunologically, physiologically, orgenetically distinct from the primordial state.

As described above, the present invention provides methods of adaptingplants and animals to survive in a hostile environment, wherein themethod comprises culturing undifferentiated cells from the plant oranimal in a weightless condition that mimics at least one element of thehostile environment to which the animal or plant is to be adapted;selecting the cells that proliferate in said condition; and cultivatingsaid selected cells to produce plants and animals that are adapted togrow in that particular hostile environment.

Any suitable means for achieving reduced gravity or microgravityconditions can be used for performing the method. In one embodiment, themethod is performed under reduced gravity or microgravity conditions inspace, e.g., aboard the Space Shuttle, the Space Station, a soundingrocket, or a satellite. In another embodiment, the method is performedunder reduced gravity or microgravity conditions simulated on Earthusing a machine or other device suitable for this purpose.

As used herein, the term “hostile environment” is used interchangeablywith “non-natural environment” and means an environment in which theplant or animal does not normally exist or survive. By way of example, ahostile environment for a banana plant would be the Arctic Circle.Another example of a hostile or non-natural environment for almost anyplant or animal would be the surface of Mars.

Many elements of the particular hostile environment of interest can bechosen as the selection pressure to “force” the undifferentiated cellsfrom the plant or animal to express a subset of genes that will enablethem to adapt to the hostile environment. As used herein, the term“force” means to apply a selection pressure to the population ofproliferating undifferentiated cells to obtain cells that survive in thecondition of interest. Some environmental elements suitable for useinclude, but are not limited to, temperature, such as excessive heat orexcessive cold, high or low concentrations of carbon dioxide, barometricpressure, radiation levels, humidity levels, oxygen concentration, lowsunlight exposure, extreme drought, extreme salinity, and the presenceof environmental toxins.

In one embodiment, the present invention provides a method of adapting aplant to grow in a hostile environment, wherein the method comprisesculturing undifferentiated cells from the plant in a weightlesscondition that mimics at least one element of the hostile environment towhich the plant is to be adapted; selecting the cells that proliferatein said condition; and cultivating said selected cells to produce matureplants, wherein the mature plants are adapted to grow in said hostileenvironment. The method can be similarly applied to adapting an animalto grow in a hostile environment.

In one embodiment, the method further comprises evaluating the matureplants in the hostile environment. The plants may be evaluated onseveral criteria including, but not limited to, length of survival,growth rate, reproductive capability, cell structure, and geneexpression. Further, the undifferentiated cells that are cultured can beobtained from uniting a pollen and ovule of a plant or a sperm and eggof an animal prior to culturing in a weightless condition or environmentor alternatively, can be united in a gravity condition and preserved tobe cultured in a weightless environment.

In one embodiment, plants suitable for use in the methods of presentinvention include dicotyledons. In certain exemplary embodiments, thedicotyledons may include leguminous plants and other large seed dicots,e.g., peanuts, soybeans, common beans, squash, zucchini, peppers,melons, cucumbers and others. Other dicots for use in the inventioninclude potatoes, tomatoes, alfalfa, canola, apples, and pairs. Incertain other embodiments, a plant suitable for use in the invention canbe a woody dicot, including pome fruits, citrus crops, and vegetablecrops.

In other embodiments, plants suitable for use in the methods of presentinvention include be monocotyledons. In certain exemplary embodiments,the monocotyledons include may include corn (“maize”), rice, wheat,barley, sorghum, rye, banana, plantains, and other grasses.

In another embodiment, the plant may be from the genus Jatropha.Jatropha is a genus of approximately 175 succulent plants, shrubs andtrees (some are deciduous, like Jatropha curcas L.), from the familyEuphorbiaceae. The hardy Jatropha is resistant to drought and pests, andproduces seeds containing up to 40% oil. When the seeds are crushed andprocessed, the resulting oil can be used in a standard diesel engine,while the residue can also be processed into biomass to powerelectricity plants and jet engines. Thus, Jatropha curcas is a promisingcandidate for future biofuel and energy production. Therefore, expandingthe range of habitats in which it can survive is of great interest andimportance.

In another embodiment, the organism may be a lichen. Lichens arecomposite organisms consisting of a symbiotic association of a fungus(the mycobiont) with a photosynthetic partner (the photobiont orphycobiont), usually either a green algae or cyanobacterium. Themorphology, physiology and biochemistry of lichens are very different tothat of the isolated fungus and alga in culture. Lichens occur in someof the most extreme environments on Earth—arctic tundra, hot deserts,rain forests, rocky coasts and toxic slag heaps.

In another embodiment, the organism may be an algae or fungus bythemselves. In other embodiments, the algae or fungus may be associatedwith other primitive organisms, such as lower plants, including, but notlimited to, Thallophytes, Chlorophyceae (for example, green algae,spirogyra, or vaucheria) and Phycomycetes (for example, algae fungi,bread mold, or water mold).

In another embodiment, the undifferentiated cells may be from a plantthat has been genetically modified to result in a specific phenotype.There are numerous examples of plants that have been transformed withspecific genes so that the resulting transgenic plants exhibit aparticular characteristic, such as resistance to a particular pathogenor increased size of fruit. For instance, herbicide resistant plants asdisclosed in U.S. Pat. No. 7,169,970, plants that have enhanced nitrogenassimilation as disclosed in U.S. Pat. No. 6,107,547, and tomatoes witha delayed ripening phenotype as disclosed in U.S. Pat. No. 5,952,546 arejust a few of the various examples of genetically-modified plants thathave been created. Undifferentiated cells may be obtained from any ofthe many varieties of transgenic plants for use in the methods of thepresent invention.

In some embodiments, certain plant species known to be suitable for useas biofuels may be modified, tailored, altered, enhanced, by on-orbit,weightless processing of the cells and/or cellular components, such thathigher-energy by weight or by volume biofuel product may be producedfrom said plant seeds and cultivars, and that these species may includethe known, 1st, 2nd, 3rd, and 4th generation biofuels, including but notlimited to corn, soy, sugar cane, sugar beet, sweet sorghum, maize,palm, pinnata, switchgrass, rapeseed, miscanthus, hemp and other knownsuitable biofuels of the 1st generation, as well as Jatropha (asdescribed above), Camelina, Manihot, and algae, including in particularmarine algae, as third generation biofuels, including halophytes,particularly Salicornia. This embodiment further contemplatesmodifications, alterations, and optimizations of these biofuel speciesas a result of weightless (microgravity) cellular processes executed inthe zero-force weightless environment on-orbit, as claimed herein, thatenable and result in the production of useful and valuable geneticallyaltered and tailored biofuel and crop outputs and end-products.

Methods of obtaining undifferentiated plant cells are well known in theart. A mass of undifferentiated plant cells may be obtained byaseptically removing a small piece of plant tissue from a selectedorgan, such as from the root, stem, etc., and placing it in a sterilemedium containing appropriate nutrients. Such a tissue explant will growand proliferate into a large number of the same type of plant cells orof related plant cells, without specialization of these cells to formspecific plant organs such as roots or leaves, etc. These cells may bereferred to as a heterogeneous population or colony of undifferentiatedplant cells comprised of single cells as well as aggregates of cells.This type of uninterrupted cell growth and multiplication without theformation of specific plant organs is known as undifferentiated cellgrowth.

In one embodiment, the undifferentiated plant cells to be used inmethods of the invention may be obtained from the undifferentiatedparenchyma from the apical meristems of the plant. Reproduction and useof apical cell reproduction has greatly increased the numbers of plantsin a vegetative reproduction process. The process depends on theisolation of the reproducing cells at the tip of a plant or plant part(root, branch, etc.) known as the meristem and successful cloning of thelimited number of cells at the undifferentiated stage of development atthe tip of the plant or other actively growing portions of the plant(root, cambium, etc.). Suspension cultures of undifferentiated cells maybe prepared from meristem isolates.

Alternatively, the undifferentiated plant cells suitable for use in themethods of the invention may be obtained by proliferation or replicationof diploid cells formed by the union of pollen (sperm) and ovule (egg)from the particular plant species of interest under weightlessconditions as described herein. It is possible to replicate and produceundifferentiated parenchyma resulting from the unification of pollen(sperm) and egg (ovary) in plants that are unified on Earth, preservedprior to any division of the united single cell, and transportedimmediately to orbit for the purpose of producing undifferentiated cellscapable of replicating identical cells for production of tissues usedfor parts of plants, and the plant itself, including, but not limited tostems, roots, flowers, seeds, fruits, and other tissues. The union ofpollen and ovary (egg) in zero gravity will produce a cell that will goto mitosis and then reproduce that cell continually en masse or until agenetic break down in the cell(s) may occur that would disrupt theexponential reproduction of the same mitosis. The newly formed cells cansubsequently be used in the methods of the present invention.

The following example outlines an experiment for adapting a species ofcitrus plant to grow in colder climates. This example is forillustration purposes only and in no way limits the scope of theinvention. As described above, various types of plants may be used inthe methods of the invention. Similarly, many different elements of ahostile environment may be used as selective pressures to adapt theplants.

EXAMPLE Method of Adapting a Citrus Plant to Thrive in Cold Climates

Suspension cultures have been widely used for tissue culture and massclonal propagation of a diverse array of higher plants, and also asmodels for studies of cell development and differentiation. Analysis ofthese suspension cultures determine structural and genetic changes inundifferentiated plant cells submitted to the effects of environmentalelements, such as abnormal temperatures. In addition, cell growth andreplication are assessed visually. Structural changes are performedthrough histological analyses, including light microscopy, transmissionelectron microscopy (TEM), and if feasible, scanning electron microscopy(SEM). Genetic analyses is performed to evaluate differential geneexpression under the specific environmental condition.

Cell suspension cultures are initiated for a variety of citrus tree(e.g. Citrus sinensis) that has superior fruit, but is not cold tolerantbelow 28° F. Cultures are prepared by excising the undifferentiatedparenchyma cells from the apical meristems of the plant about one daybefore space shuttle launch. The cell suspensions are cultured on MSmedium modified with 1 mg/L 2,4-D. Once a significant amount of cellsare produced, they are transferred to 10-ml opticells and cultured asdescribed above.

On orbit, in the opticells the experimental cell suspension cultures aresubjected to temperatures of 25° F. for a predetermined period of time,such as for several weeks or about three months or more on theInternational Space Station (ISS). Also on orbit, corresponding controlcell suspension cultures would be exposed to the optimal growingtemperature for that species of citrus. Cells exposed to each growingtemperature would be returned to Earth and a portion used for furtheranalyses (see below). The other portion would immediately be separatedinto individual cell containers with agar and cultured to determinewhich cells survived. The cells that survived can be nurtured to maturetrees and then subjected to temperatures of 25° F. to determine thelevel of cold tolerance achieved. Other parameters of the mature treeswould also be measured, including yield of the trees, length ofsurvival, and growth.

Samples from suspension cells maintained in opticells, under bothexperimental (25° F.) and control (greater than 28° F.) temperatureconditions are collected and compared for histological and geneticanalysis. For histological analyses, cell suspensions are prepared forlight and electron microscopy. Opticells are compatible for use withstandard, phase contrast, confocal, and high-resolution time-lapse videomicroscopes. Cells are examined microscopically on either opticellgrowth surface or in between. Oil immersion lenses (up to 100×) are usedon the membrane without disruption or contamination. The membrane issectioned for small scale staining and microscopy. Additional samplesare removed and fixed in glutaraldehyde for subsequent evaluation ofcell ultrastructure through TEM and SEM.

By the term “control” or “control cells” or “microorganisms” within themeaning of the present invention, is meant cells or microorganisms grownon earth or in a gravity environment as compared to the cells ormicroorganisms grown in a weightless or microgravity environment asdescribed herein or on the Space Station. Additionally, control orcontrol cells or microorganisms can also mean those cells ormicroorganisms grown under a normal non-stressed environment, ascompared to the stress environment factors and stimuli, as set forthherein.

Gene expression analyses are performed to evaluate possible genes thatare either up-regulated or down-regulated in response to the coldertemperature. Suspension cultures maintained in space are fixed inRNAlater (Ambion) liquid preservative through the Kennedy Space Centerfixation tube (UT), hardware designed to provide proper containment offixatives for biological samples in space placed inside the C-habenvironment. RNA is isolated and compared for suspension cultures inboth temperature conditions to evaluate gene expression. Molecularbiology techniques for reverse transcriptase polymerase chain reaction(RT-PCR) and/or copy-DNA amplified fragment length polymorphism(cDNA-AFLP) and gel electrophoresis are performed according towell-known techniques to those skilled in the art and are used for geneexpression analyses. Microarray analysis of gene expression isperformed. Results of the microarray data identify the genes involved inthe tolerance factor for cold. Genes involved in cold-toleranceadaptation will typically show at least a four-fold change in expressioncompared to the control cells exposed to the normal growing temperature.

Additional evaluations of the cell suspensions may also be conductedincluding, but not limited to, cell growth rates, cell densities,subculture frequency, and size and condition of cells.

The above-described techniques are also applicable to adapting animalsto hostile environments. In certain embodiments, the animal is a mammal.As used herein, the term “mammal” refers to any mammal. Nonexclusiveexamples of such mammals include, but are not limited to, animals suchas dogs, cats, horses, cattle, sheep, and goats. In other embodiments,the animal may be a bird. In yet other embodiments, the animal may be anaquatic species.

In one embodiment, the invention provides a method of adapting an animalto grow in a hostile environment, wherein an element of the hostileenvironment is selected from the group consisting of heat, cold,barometric pressure, excessive radiation, high carbon dioxide levels,low humidity, high humidity, chemical pollutants, disease, extremesalinity, reduced or increased exposure to sunlight, low water anddrought conditions, excess water conditions or combinations thereof,including high and low levels or concentrations of any of the describedconditions.

In some embodiments, the undifferentiated cells from animals suitablefor use in the methods of the invention can be embryonic stem cells.Methods for isolating embryonic stem cells are well known to those ofskill in the art, including, but not limited to, somatic nucleartransfer, cell fusion, and genetic manipulation techniques that createtotipotent cells that are capable of generating all the tissues of theentire animal.

Alternatively, the undifferentiated animal cells can be obtained bymethods comprising forming a diploid cell by uniting two haploid cellsand proliferating the diploid cell in a weightless condition, whereinthe diploid cell replicates itself but does not differentiate intospecialized cells and tissues. More specifically, the egg and sperm areunited using standard (IVF) techniques as described herein.

Culture conditions and media for culturing the undifferentiated cellsaccording to the methods of the invention are well known to the skilledartisan as described above.

Various methods for culturing stem cells, e.g., embryonic stem cells(ESCs), may be used with the present invention. Typically, ESCs aregrown in adherent culture systems such as on tissue culture plates. Incertain aspects, culture plates for use in the invention may comprise agel matrix such as a collagen or hydrogel matrix (e.g., a MATRIGEL™). Invarious embodiments, culture plates may be coated with, e.g., collagenIV, fibronectin, laminin, and vitronectin in combination may be used toprovide a solid support for embryonic cell culturing and maintenance, asdescribed in Ludwig et al. (2006). Matrix components which may be usedwith the present invention to coat tissue culture plates includes acollagen such as collagen IV, laminin, vitronectin, Matrigel™, gelatin,polylysine, thrombospondin (e.g., TSP-1, -2, -3, -4 and/or -5), and/orProNectin-F™. Three dimensional support matrices for use in tissueculture have been previously described for example in U.S. PublicationNos. 20060198827 and 20060210596, each incorporated herein by reference.The skilled artisan will recognize that in certain aspects adherenttissue culture cells may be defined by the cell density or confluency.Thus, in some cases, methods of the invention involve expansion ofproliferating cells from a high density to a lower density to facilitatefurther cell proliferation. For example, methods for expanding cellsaccording to the invention may involve a first population of embryonicstem (ES) cells that is between about 50% and 99% confluent. Forexample, in certain aspects the first population of ES cells may beabout or less than about 60%, 70%, 80%, 90% or 95% confluent.Furthermore, in certain aspects expansion or passage of adherent EScells may involve seeding separated cells in fresh growth media. As usedherein the term “seeding” cells means dispersing cells in growth mediasuch that the resultant cell culture(s) are of approximately uniformdensity. Thus, seeding of cells may involve mixing separated cells withfresh growth media and/or spatially dispersing separated cells over thesurface of a tissue culture plate.

Undifferentiated propagation of adherent colonies of ESCs may beaccomplished with a Knockout (KO) serum-free culture system without theuse of feeders by plating and growing the colonies on extracellularmatrices (ECM) within a feeder-conditioned KO-DMEM medium supplementedwith KOSR and fibroblast growth factor 2 (FGF2). Media available fromcommercial sources, such as Gibco Invitrogen Corporation, Grand Island,N.Y. Furthermore, it has been suggested that feeder conditioning may bereplaced by substituting the medium with high concentrations of FGF2 andnoggin. Alternatively, feeder conditioning was replaced by transforminggrowth factor-1 and human leukemia inhibitory factor (LIF) (in additionto FGF2) and growing the cells on human fibronectin, or by serum-freemedia supplemented with soluble factors including FGF2, activin A,transforming growth factor-β1 (TGF-(β1), pipecolic acid, GABA, LiCL andculturing the cells on ECM components. In general, a key limitation ofESC culture systems is that they do not allow the propagation of purepopulations of undifferentiated stem cells and their use typicallyinvolves some level of background differentiation. The stem cells mostcommonly follow a default pathway of differentiation into an epithelialcell type that grows either as a monolayer of flat squamous cells orform cystic structures. Most probably, this form of differentiationrepresents differentiation of human ESC (hESC) into extraembryonicendoderm.

In these adherent culture systems of colonies, the ESCs are mostcommonly propagated (mechanically and/or by using enzymatic digestion)as clusters, on a small scale. These culture systems arelabor-intensive, highly variable, may contain undefined factors, and donot provide steady-state operating conditions. Most importantly, they donot typically allow for large scale production of standardizedhomogenous undifferentiated ESCs needed for the aforementioned uses.

Suspension culture bioreactors offer several advantages over theconventional use of static monolayer cultures. These systems facilitatethe large-scale expansion of the cells in a homogeneous cultureenvironment, thus decreasing the risk of culture variability. They arealso less labor-intensive to operate and offer the possibility ofcomputer control and monitoring of the culture conditions. Althoughbioreactors have been used to expand neural stem cells, mouse ES cellsand differentiating hESCs within embryoid bodies (EBs), only recentlysome progress has been made towards the development of protocols for thefeeder-free expansion of undifferentiated hESCs in suspension systems(see US20070212777, or J. Biotechnology, Vol. 132 (2), 227-236 (2007),which are herein incorporated by reference in its entirety).

The present invention also provides methods of identifying genesassociated with adaptation of a plant or animal to a hostileenvironment. Previous experiments demonstrated that seedlings fromArabidopsis thaliana grown under microgravity (weightless condition)exhibited a change in expression of select genes compared to theircounterpart controls grown on Earth. The microarray data (FIG. 3) fromthese experiments showed that 182 genes were differentially expressedwith at least a four-fold change in expression. Some of thedifferentially expressed genes were identified as heat-shock and/or CAB(light) genes. Using the same principles on which these experiments arebased, the present invention provides methods for identifying specificgenes differentially expressed between a control condition and aparticular environmental condition. These identified genes may play arole in the adaptation of the plant or animal to that particularenvironment.

In one embodiment of the invention, the method comprises culturingundifferentiated cells from a plant or animal in a weightless conditionthat mimics at least one element of the hostile environment to which theplant or animal is to be adapted; selecting the cells that proliferatein said condition; and examining the gene expression profile of theselected cells in comparison to the gene expression profile of controlcells; and identifying genes that have a change in expression level,wherein the identified genes are associated with adaptation to thehostile environment.

The change in expression level is at least 2-fold, at least 4-fold, atleast 6-fold, at least 10-fold, at least 15-fold, or at least 20-fold.The change in expression level could be an increase in expression or adecrease in expression. Thus, particular environmental stimuli mayproduce both up-regulation and down-regulation of specific genes.

In one embodiment, the selected genes that are differentially expressedin the various environmental conditions can be further used to producetransgenic plants and animals with the desired adaptive characteristicsby introducing these genes into cells that mature into plants oranimals.

Therefore, the present invention further encompasses a plant or animalor undifferentiated cell thereof produced by the methods describedherein, wherein said plant, animal or undifferentiated cell thereofcomprises at least one identified gene that has a change in expressionlevel as compared to the gene expression profile of control cell,wherein the identified genes are associated with adaptation to thehostile environment.

In one embodiment, a transgenic plant tolerant to environmentalstresses, such as low temperature, freezing, and dehydration stresses,can be produced by introducing DNA encoding the protein of the interestinto a host plant using genetic engineering techniques. Methods forintroducing the gene into a host plant include indirect introductionsuch as the Agrobacterium infection method and direct introduction suchas the particle gun method, polyethylene glycol method, liposome method,and microinjection method.

In the present invention, while the host for the transformant is notparticularly limited, it is preferably a plant. The plant may be anycultured plant cells, the entire plant body of a cultured plant, plantorgans (such as leaves, petals, stems, roots, rhizomes, or seeds), orplant tissues (such as epidermis, phloem, parenchyma, xylem, or vascularbundle). Plants may be monocotyledonous plants such as rice, maize, andwheat. When a cultured plant cell, plant body, plant organ or planttissue is used as the host, the Agrobacterium infection method, particlegun method, or polyethylene glycol method can be employed to introducethe DNA encoding the protein of the present invention to transform thishost plant by introducing a vector into plant sections. Alternatively, avector can be introduced into a protoplast by electroporation to producea transformed plant.

For example, when a gene is introduced into Arabidopsis thaliana by theAgrobacterium infection method, the step of infecting the plant with anAgrobacterium containing a plasmid comprising the gene of interest isessential. This step can be performed by the vacuum infiltration method[CR Acad. Sci. Paris, Life Science, 316:1194 (1993)]. Specifically,Arabidopsis thaliana is grown in a soil composed of equivalent portionsof vermiculite and perlite. The Arabidopsis thaliana is immerseddirectly in a culture fluid of an Agrobacterium, containing a plasmidcomprising the gene of interest, placed in a desiccator, and then suckedwith a vacuum pump to 65-70 mmHg. Then, the plant is allowed to stand atroom temperature for 5-10 min. The plant pot is transferred to a tray,which is covered with a wrap to maintain humidity. On the next day, thewrap is removed. The plant is grown in that state to harvest seeds.

Subsequently, the seeds are sown on MS agar medium supplemented withappropriate antibiotics to select those individuals which have the geneof interest. Arabidopsis thaliana grown on this medium are transferredto pots and grown there. As a result, seeds of a transgenic plant intowhich the gene of the interest has been introduced can be obtained.Generally, the genes are introduced into the genome of the host plant ina similar manner. However, due to differences in the specific locationson the genome into which the genes have been introduced, the expressionof the introduced genes varies. This phenomenon is called “positioneffect.” By assaying transformants with DNA fragments from theintroduced gene as a probe by Northern blotting, it is possible toselect those transformants in which the introduced gene is expressedmore highly.

The confirmation that the gene of interest is integrated in thetransgenic plant into which the gene of the present invention has beenintroduced and in the subsequent generation thereof can be made byextracting DNA from cells and tissues of those plants and detecting theintroduced gene by PCR or Southern analysis, which are conventionalmethods in the art.

The expression level and expression site of a gene in a transgenic plantinto which the gene of the present invention has been introduced can beanalyzed by extracting RNA from cells and tissues of the plant anddetecting the mRNA of the introduced gene by RT-PCR or Northernanalysis, which are conventional methods in the art. Alternatively, theexpression level and expression site can be analyzed directly by Westernblotting or the like of the gene product of the present invention usingan antibody against the above product.

In one embodiment, the gene of interest encodes a transcription factor.Because stress responses such as drought tolerance involve coordinatedchanges in many genes, the ability to affect many changes with one geneis an attractive proposition. Transcription factors can activatecascades of genes that function together to enhance stress tolerance.Transcription factors refer to a class of genes that control the degreeto which other genes in a cell are activated. Transcription factors areable to recognize and bind to regions of DNA that have a specificsequence in the promoters of the genes they regulate. Thus, if a dozengenes all have that region of DNA somewhere in their promoters, theywill all be regulated by the same transcription factor. Becausetranscription factors are key controlling elements of biologicalpathways, altering the expression levels of one or more transcriptionfactors can control a variety of genes involved in the stress response.

In another embodiment, the gene of interest encodes a stress-inducedprotein. In an exemplary embodiment, the gene of interest encodes aheat-shock protein.

The control cells of the present invention to which the experimentalcells are compared would include undifferentiated cells proliferating ina weightless condition exposed to the normal or native environmentalelement of that which is being varied in the experimental condition. Forexample, in a method to adapt a plant to grow in arid conditions, theundifferentiated cells from the plant would be exposed to a low humidityenvironment in the experimental condition and an environment havingnormal humidity for that particular plant species in the controlcondition.

In another embodiment, the method can be used for the production ofvaccines to be used in animals and humans. Strains of Salmonella onorbit have previously been shown to demonstrate increases in virulence.See Wilson et al., PLoS One 12(3): e3923: 1-10.

In one embodiment, the invention provides a method of adapting apathogenic microorganism to grow in a hostile, non-native environmentcomprising culturing the microorganism in a weightless condition thatmimics at least one element of the hostile environment to which themicroorganism is to be adapted; selecting the microorganisms thatreplicate in said condition; cultivating said selected microorganism toproduce a modified strain of microorganism, wherein the microorganism isare adapted to grow in said hostile environment; and optionallyevaluating the microorganism to determine the properties of saidmodified microorganism.

In another embodiment, the virulence of the modified microorganism isdifferent than the virulence of the original non-adapted microorganism.

The technology described in this invention can be used to “force” theexpression of genes on orbit to produced microorganisms, such as abacteria or virus, with desired properties for vaccine production. Bystressing, for example, a pathogenic microorganisms on orbit, modifiedstrains of microorganisms can be produced. These modified microorganismscan include less virulent and/or more virulent strains of bacteria andviruses, which can then be utilized for the production of improvedvaccines. The vaccines produced by this method can be applied totreatment and/or prevention of many animal and human diseases,including, but not limited to, hoof and mouth disease and brucellosis.

The foregoing detailed description has been given for clearness ofunderstanding only and no unnecessary limitations should be understoodtherefrom as modifications will be obvious to those skilled in the art.

It is understood that the present invention is not limited to theparticular methods and components, etc., described herein, as these mayvary. It is also to be understood that the terminology used herein isused for the purpose of describing particular embodiments only, and isnot intended to limit the scope of the present invention. It must benoted that as used herein, the singular forms “a,” “an,” and “the”include the plural reference unless the context clearly dictatesotherwise.

All publications, patents, and patent applications identified above areincorporated herein by reference in their entirety. Although thisinvention has been described in relation to certain specific embodimentsthereof, and many details have been set forth for purposes ofillustration, it will be apparent to those skilled in the art that theinvention is susceptible to additional embodiments and that certain ofthe details described herein may be varied considerably withoutdeparting from the basic principles of the invention.

What is claimed is:
 1. A method of adapting a plant to grow in a hostileenvironment, the method comprising: providing replicating parenchymacells to produce a cell suspension culture comprising a plurality ofundifferentiated parenchyma cells; culturing the cell suspension culturewhile simultaneously exposing the cell suspension culture to both amicrogravity condition and a stimuli corresponding to the hostileenvironment to which the plant is to be adapted; and harvesting cellsthat replicate during the step of culturing, wherein the culturingsimultaneously exposes the cell suspension culture to both themicrogravity condition and the stimuli corresponding to the hostileenvironment to cause the plurality of undifferentiated parenchyma cellsto express genes that enable the harvested cells to adapt to the hostileenvironment; and sequentially removing the harvested cells from themicrogravity condition and cultivating the harvested cells to produce amature plant.
 2. The method of claim 1 further comprising evaluatingsaid mature plant.
 3. The method of claim 2, wherein said mature plantis evaluated for at least one of length of survival, growth rate,reproductive capability, cell structure, gene expression, orcombinations thereof.
 4. The method of claim 1, wherein the parenchymacells are obtained from uniting a pollen and ovule of a plant prior toexposure to the microgravity condition.
 5. The method of claim 1,wherein the hostile environment is selected from the group consisting ofheat, cold, low barometric pressure, excessive radiation, high carbondioxide levels, low oxygen levels, low humidity, high humidity, extremesalinity, reduced or increased exposure to sunlight, and low waterconditions.
 6. The method of claim 1, wherein the parenchyma cells areobtained by uniting a pollen and an ovule in the microgravity condition,wherein said parenchyma cells obtained from said union do not developinto differentiated cells.
 7. The method of claim 1, further comprisingsteps of: examining a gene expression profile of the harvested cells incomparison to the gene expression profile of control cells; andidentifying genes that have a change in expression level as compared tothe gene expression profile of the control cells; wherein saididentified genes are associated with adaptation to the hostileenvironment.
 8. The method of claim 7, wherein the change in expressionlevel is at least four fold in comparison to the expression profile ofthe control cells.
 9. A method of adapting a plant to grow in a coldenvironment, the method comprising: providing replicating parenchymacells to produce a cell suspension culture comprising a plurality ofundifferentiated parenchyma cells; culturing the cell suspension culturecomprising the plurality of undifferentiated parenchyma cells whilesimultaneously exposing the cell culture to both a microgravitycondition and a cold environment to which the plant is to be adapted;and harvesting cells that replicate during the step of culturing,wherein the culturing simultaneously exposes the cell suspension cultureto both the microgravity condition and the cold environment, to causethe plurality of undifferentiated parenchyma cells to express genes thatenable the harvested cells to adapt to the cold environment, where theplant is not a citrus plant.
 10. The method as recited in claim 9,wherein the cold environment is a temperature of about 25° F. or less.11. The method of claim 10, further comprising the sequential step of(1) removing the harvested cells from the microgravity condition and (2)cultivating the harvested cells to produce a mature plant.
 12. A methodof adapting a plant to grow in a hostile environment, the methodcomprising: providing replicating parenchyma cells to produce cellsuspension culture comprising a plurality of undifferentiated parenchymacells; culturing the cell suspension culture comprising the plurality ofundifferentiated parenchyma cells while simultaneously exposing the cellculture to both a microgravity condition and a hostile environment towhich the plant is to be adapted, wherein the hostile environment isselected from the group consisting of heat, cold, low barometricpressure, excessive radiation, high carbon dioxide levels, low oxygenlevels, low humidity, high humidity, extreme salinity, reduced orincreased exposure to sunlight, and low water conditions; and harvestingcells that replicate during the step of culturing, wherein the culturingsimultaneously exposes the cell culture to both the microgravitycondition and the hostile environment to cause the plurality ofundifferentiated parenchyma cells to express genes that enable theharvested cells to adapt to the hostile environment, wherein saidharvested cells do not develop into differentiated cells during the stepof culturing; sequentially removing the harvested cells from themicrogravity condition and cultivating the harvested cells to produce amature plant.
 13. The method of claim 12, further comprising a step ofexcising the parenchyma cells from a cellular sample, the step ofexcising occurring prior to the step of replicating.